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Systems Biology and Biomarkers of Early Effects for Occupational Exposure Limit Setting
In a recent National Research Council document, new strategies for risk assessment were described to enable more accurate and quicker assessments.(1)This report suggested that evaluating individual responses through increased use of biomonitoring could improve dose-response estimations. Identification of specific biomarkers may be useful for diagnostics or risk prediction as they have the potential to improve exposure assessments. This paper discusses systems biology, biomarkers of effect, and computational toxicology approaches and their relevance to the occupational exposure limit setting process.The systems biology approach evaluates the integration of biological processes and how disruption of these processes by chemicals or other hazards affects disease outcomes. This type of approach could provide information used in delineating the mode of action of the response or toxicity, and may be useful to define the low adverse and no adverse effect levels. Biomarkers of effect are changes measured in biological systems and are considered to be preclinical in nature. Advances in computational methods and experimental -omics methods that allow the simultaneous measurement of families of macromolecules such as DNA, RNA, and proteins in a single analysis have made these systems approaches feasible for broad application.The utility of the information for risk assessments from -omics approaches has shown promise and can provide information on mode of action and dose-response relationships. As these techniques evolve, estimation of internal dose and response biomarkers will be a critical test of these new technologies for application in risk assessment strategies. While proof of concept studies have been conducted that provide evidence of their value, challenges with standardization and harmonization still need to be overcome before these methods are used routinely.
In a recent National Research Council document, new strategies for risk assessment were described to enable more accurate and quicker assessments.This report suggested that evaluating individual responses through increased use of biomonitoring could improve dose-response estimations. Identification of specific biomarkers may be useful for diagnostics or risk prediction as they have the potential to improve exposure assessments. This paper discusses systems biology, biomarkers of effect, and computational toxicology approaches and their relevance to the occupational exposure limit setting process.
The systems biology approach evaluates the integration of biological processes and how disruption of these processes by chemicals or other hazards affects disease outcomes. This type of approach could provide information used in delineating the mode of action of the response or toxicity, and may be useful to define the low adverse and no adverse effect levels. Biomarkers of effect are changes measured in biological systems and are considered to be preclinical in nature. Advances in computational methods and experimental -omics methods that allow the simultaneous measurement of families of macromolecules such as DNA, RNA, and proteins in a single analysis have made these systems approaches feasible for broad application.
The utility of the information for risk assessments from -omics approaches has shown promise and can provide information on mode of action and dose-response relationships. As these techniques evolve, estimation of internal dose and response biomarkers will be a critical test of these new technologies for application in risk assessment strategies. While proof of concept studies have been conducted that provide evidence of their value, challenges with standardization and harmonization still need to be overcome before these methods are used routinely.
# Introduction
C urrently, little toxicity data exist for most of the 82,000 chemicals used in the United States, which greatly hampers risk assessment and management activities.In addition, it is rare for workers or the general public to be exposed to only to a single compound, but rather they are exposed to complex mixtures that may have additive, synergistic, or antagonistic actions. The complexity of exposure scenarios and lack of data make risk management decisions difficult and time consuming.
The systems biology approach is based on consideration of normal biological processes (including pathways leading to effects and homeostatic and adaptive responses) and how chemicals disrupt those processes.This type of approach would provide integrated information that could be used in delineating the mode(s) of action (MOA) of the adverse response or toxicity. [bib_ref] Toxicogenomic profiling of chemically exposed humans in risk assessment, Mchale [/bib_ref] Different doses can produce widely different responses in an organism. Some of the responses are of no consequence to the health or viability of the organism, others may be beneficial (e.g., antioxidant), and others are toxic. [bib_ref] Dose response considerations in risk assessment-an overview of recent ILSI activities, Holsapple [/bib_ref] Biomarkers have been defined by the National Academy of Sciences (NAS) as measurable changes in a biological system or organism or measured alterations in structure or function .Biomarkers may be indicative of exposure, response or effect, and susceptibility and can be used to monitor exposures and a wide variety of responses ranging from abnormal development to early disease indicators. [bib_ref] Perera: Validation, Schulte [/bib_ref] Occupational exposures are mainly by inhalation or through the dermal route, while the primary route for general environmental exposures is by ingestion. Route of exposure may affect the level of internal dose and therefore the toxicity. Biomarker measurements are an aggregate of all exposure pathways. One benefit of early response biomarkers is in their interpretation within the context of integrated systems models, which connect these biomarkers to adverse outcomes of regulatory concern. Advances in computational methods [bib_ref] Molecular networks as sensors and drivers of common human diseases, Schadt [/bib_ref] and experimentalomics methods that allow the simultaneous measurement of families of macromolecules such as DNA, RNA, and proteins in a single analysis [bib_ref] Toxicology and genetic toxicology in the new era of "toxicogenomics": Impact of..., Aardema [/bib_ref] have made these systems approaches feasible for broad application in both pharmaceutical discovery [bib_ref] Molecular networks as sensors and drivers of common human diseases, Schadt [/bib_ref] and environmental risk assessment. [bib_ref] Systems biology and mode of action based risk assessment, Edwards [/bib_ref] The promise is that information on hazard characterization, dose response, and risk characterization can be generated by -omics methods and used in risk assessments. [bib_ref] Temporal concordance between apical and transcriptional points of departure for chemical risk..., Thomas [/bib_ref] This article focuses on systems biology, biomarkers of effect/response, and computational toxicology approaches and their relevance to the occupational exposure limit (OEL) setting process. A glossary of key terms relating to this topic is provided in [fig_ref] TABLE I: Glossary of Key Terms [/fig_ref].
Key points of emphasis covered in this article include the following.
- Many drivers exist for increased reliance on systems biology approaches that are pushing changes in health risk assessment methods, including for OEL setting. - Practical long-term implications of such approaches are many, including decreased uncertainty in OELs through improved understanding of biological responses at lower levels of chemical exposure.
## Figure 1.
Continuum from exposure to disease. Adapted from NRCand Schulte and Perera. [bib_ref] Perera: Validation, Schulte [/bib_ref]. Reproduced from Environmental Health Perspectives.
- Current methods and tools for consideration of effect biomarkers and the relationship with the toxic MOA within the framework of systems biology are being used and applied in OEL setting via proof of concept studies.
## Current efforts on early response biomarkers and risk assessment
S ome efforts have been initiated in the global community to revise the way that risk assessments are conducted or to speed the data flow into risk assessments [fig_ref] TABLE I: Glossary of Key Terms [/fig_ref]. The European Registration, Evaluation, Authorisation, and Restriction of Chemicals (REACH) program seeks to determine the risk of thousands of chemicals that are produced in quantities greater than 10 tons/year. [bib_ref] Human biomonitoring as a pragmatic tool to support health risk management of..., Boogaard [/bib_ref] Derived No Effect Levels are required for all chemicals that are classified as a health hazard. Traditional toxicity testing is unsustainable and unethical under this paradigm, because of the large number of animals that would be needed and the associated high cost. [bib_ref] An animal protection perspective on 21st century toxicology, Stephens [/bib_ref] Efforts to establish a sustainable strategy for toxicity testing in the United States were accelerated when the National Research Council (NRC) defined a vision of toxicity testing in the 21 st century that called for greater use of in vitro testing, computational system approaches, and a reduction of expensive animal testing. [bib_ref] Toxicity testing in the 21st century: bringing the vision to life, Andersen [/bib_ref] In the NRC strategy, toxicity testing would evaluate specific perturbations in identified pathways rather than by direct evidence of adverse effects; therefore, risk assessments would be revised to incorporate this new information.These new technologies could be performed faster and cheaper and evaluate toxicity of a larger number of concentrations.
Shortly after the NRC report on toxicity testing,a separate NRC committee published recommendations on the use of toxicogenomic technologies and the need for more predictive toxicity testing for incorporation into risk assessments.Improvements in cross-species extrapolation, identification of vulnerable or sensitive populations, determination of life stage effects, investigation of mechanisms of action, and refinement of exposure assessments are all potential uses for toxicogenomic data.Computational toxicology was the subject of a National Academy of Sciences Standing Committee on Use of Emerging Science for Environmental Health Decisions meeting in September 2009. The field of computational toxicology has emerged in an effort to build predictive models from biomarker of effect data generated by omics technologies. [bib_ref] Computational toxicology: a state of the science mini review, Kavlock [/bib_ref] Computational toxicology identifies trends and patterns in biomarker and chemistry datasets. [bib_ref] Daston: Computational toxicology: realizing the promise of the toxicity testing in the..., Rusyn [/bib_ref] These models use chemical characterization to predict fate and transport as well as hazard identification. Computational toxicology also seeks to describe ways through which chemicals cause toxicity by developing computational tools that better utilize high throughput screening (HTS) and toxicogenomics data for hazard prediction. This includes models at varying levels of biological complexity, from relatively simple statistical models [bib_ref] Endocrine profiling and prioritization of environmental chemicals using ToxCast data, Reif [/bib_ref] [bib_ref] Estimating toxicityrelated biological pathway altering doses for high-throughput chemical risk assessment, Judson [/bib_ref] [bib_ref] Application of transcriptional benchmark dose values in quantitative cancer and noncancer risk..., Thomas [/bib_ref] to advanced dose-response and virtual tissue models. [bib_ref] Virtual tissues in toxicology, Shah [/bib_ref] [bib_ref] Conolly: Computational systems biology and dose-response modeling in relation to new directions..., Zhang [/bib_ref] The field of
## Key term definition
Benchmark dose A dose of a substance that when absorbed produces a predetermined change in the response rate of an adverse effect relative to the background response rate of this effect. Benchmark response (BMR)
A predetermined change in the response rate of an adverse effect relative to the background response rate of this effect. The BMR is the basis for deriving benchmark doses.Biological-based dose response models (BBDR)
A predictive model that describes biological processes at the cellular and molecular level linking the target organ dose to the adverse effect.
## Biomarkers
Internal measures or markers of exposures or effects for a chemical or agent in the body. Biomarkers of exposure
The chemical or its metabolite or the product of an interaction between a chemical and some target molecule or cell that is measured in a compartment in an organism.Biomarker of effect A measurable biochemical, physiologic, behavioral, or other alteration in an organism that, depending on the magnitude, can be recognized as associated with an established or possible health impairment or disease.Biomarker of susceptibility An indicator of an inherent or acquired ability of an organism to respond to the challenge of exposure to a specific chemical substance.
## Computational toxicology
Computational toxicology identifies trends and patterns in biomarker and chemistry datasets. [bib_ref] Daston: Computational toxicology: realizing the promise of the toxicity testing in the..., Rusyn [/bib_ref]
## Genomics
Refers to the entire genome of an organism whereas genetics is the study of a specific gene. Exposome
Concept by Wild [bib_ref] Complementing the genome with an "exposome": the outstanding challenge of environmental exposure..., Wild [/bib_ref] defined as the totality of exposure over a life time beginning in utero until death and the impact those exposures have on health. High throughput screening Experiments that can be automated and rapidly performed to measure the effect of substances on a biologic process of interest. These assays can evaluate hundreds to many thousands of chemicals over a wide concentration range to identify chemical actions on gene, pathway, and cell function. Lowest observed adverse effect level (LOAEL)
The lowest exposure level at which there are biologically significant increases in frequency or severity of adverse effects between the exposed population and its appropriate control group.
## Metabolomics
Studies the metabolic products of the human body and provides a comprehensive view of cellular metabolic changes in small molecules and byproducts. [bib_ref] Novel technologies for the discovery and quantitation of biomarkers of toxicity, Collins [/bib_ref] The metabolomics-driven approaches may provide insight into complex biochemical processes and the MOA and toxicity of chemicals. [bib_ref] Metabolomics: a global biochemical approach to drug response and disease, Kaddurah-Daouk [/bib_ref] [bib_ref] Applications of metabolomics in drug discovery and development, Wishart [/bib_ref] No observed adverse effect level (NOAEL)
The highest exposure level at which there are no biologically significant increases in the frequency or severity of adverse effects between the exposed population and its appropriate control; some effects may be produced at this dose level, but they are not considered adverse or precursors of adverse effects.(Continued on next page) Brings together toxicology, genetics, and molecular biology such as transcriptomics, proteomics, and environmental health to understand the response of an organism to an external insult. The promise of this technology is that biomarkers of exposure and effect can be elucidated. [bib_ref] Toxicogenomic profiling of chemically exposed humans in risk assessment, Mchale [/bib_ref] Insight into the mechanism of action and low-dose effects are other benefits. Transcriptomics
The study of RNA transcripts that result in gene expression.
## Uncertainty factors
A numerical value (often a factor of 3 or 10) used to adjust a point of departure (e.g., generally a no observed/lowest observed adverse effect level or benchmark dose) in order to derive a reference concentration or reference dose. Uncertainty factors are applied as needed to account for extrapolation of results in experimental animals to humans, inter-individual variability including sensitive subgroups, extrapolation from a NOAEL or LOAEL, extrapolation of results from subchronic exposures to chronic exposures, and database inadequacies.computational toxicology has rapidly expanded to include many more applications than HTS, which is still an evolving research area and in need of validation. Other applications are being utilized such as data mining the literature, in vitroin vivo extrapolations, quantitative structure activity relationships, in silico models and use of National Health and Nutrition Examination Survey biomonitoring data for identification of populations at special risk of toxicity. [bib_ref] Interpreting NHANES biomonitoring data, cadmium, Ruiz [/bib_ref] [bib_ref] Biomarkers in Toxicology and Risk Assessment, Fowler [/bib_ref] In 2009, a third NRC publication was released that examined the EPA risk assessment process and how to practically improve it to assess human health risks.Two main areas of risk analysis were evaluated, technical analysis and utility of risk. Technical analysis is how scientific information is generated and used so that more accurate risk characterizations can be obtained. Utility of risk examines the relevance of the risk assessments for making risk management decisions. A key recommendation was to improve the upfront design of risk assessments to make them more useful to answer risk management needs. In particular, the report emphasized the importance of problem formulation in determining the scope of the assessment, issues needing consideration, and options so that the risk assessment can support risk management decisionmaking. The report also noted the importance of characterizing and communicating uncertainty and variability and of placing greater emphasis on the evaluation of risk from cumulative exposure scenarios. [bib_ref] Human biomonitoring as a pragmatic tool to support health risk management of..., Boogaard [/bib_ref] Determination of risk of chemicals to improve the protection of human health and the environment National Research Council Toxicity Testing in the 21 st CenturyRecommendations for greater use of in vitro testing and computational approaches National Research Council Applications of Toxicogenomic Technologies to Predictive Toxicology and Risk AssessmentRecommendations for use of toxicogenomic technologies in risk assessment National Academy of Sciences Meeting on Use of Emerging Science for Environmental Health Decisions [bib_ref] Daston: Computational toxicology: realizing the promise of the toxicity testing in the..., Rusyn [/bib_ref] Discussion of promise of computational toxicology for policy decisions National Research Council Science and Decisions: Advancing Risk AssessmentRecommendations for improvements in the science and practice of risk assessment EPA NexGenEvaluation of use of HTS, computational toxicology and systems modeling for risk assessment
The EPA has initiated a program (http://www.epa.gov/risk/ nexgen/) to evaluate the use of HTS, computational toxicology, and systems modeling for risk assessment and risk management for environmental exposures and the general population, though not necessarily occupational exposures . [bib_ref] Advancing the next generation of health risk assessment, Cote [/bib_ref] The vision is for a tiered system that provides risk estimates on the basis of available data as well as a formal means for recommending chemicals for higher tier investigation.
## Role of biomarkers in occupational risk assessment
A major aim of biomarker research is to develop and validate biomarkers that reflect specific exposures or are quantitatively linked to adverse outcomes in humans to enable their use in risk prediction. Biomarkers have a number of advantages over "apical endpoints" typically observed in in vivo toxicology studies [fig_ref] FIGURE 2: Biologic responses as a result of an exposure [/fig_ref].
Recent advances in biomedical technology have provided powerful tools to identify new biomarkers [fig_ref] TABLE I: Glossary of Key Terms [/fig_ref]. -omics technologies are increasingly being used and have brought capabilities to investigate adverse responses, underlying toxicity mechanisms, and key toxicity pathways that have the potential to be used in risk assessment . [bib_ref] The ToxCast program for prioritizing toxicity testing of environmental chemicals, Dix [/bib_ref] [bib_ref] Empowering microarrays in the regulatory setting, Casciano [/bib_ref] [bib_ref] Future of toxicogenomics and safety signatures: balancing public access to data with..., Fostel [/bib_ref] Environmental exposures can directly or indirectly cause alterations in gene expression at either the transcriptional (gene expression) or the translational level (proteomics). Development of gene expression profiles using oligonucleotide microarrays provides a view of perturbations at the transcript level and helps identify specific genes, pathways or networks that are specific to the toxic end point of interest. [bib_ref] Exploring the new world of the genome with DNA microarrays, Brown [/bib_ref] Identifying appropriate biomarkers can be difficult because interpretation of global gene expression changes is challenging as such changes may reflect nonspecific responses or overlapping/interacting molecular processes. The use of toxicogenomics data along with other types of supportive toxicological data has been considered for hazard characterization. Recent studies have shown that benchmark dose estimates, based on gene expression omics data, for non-cancer and cancer apical endpoints can be practically applied. [bib_ref] Application of transcriptional benchmark dose values in quantitative cancer and noncancer risk..., Thomas [/bib_ref] [bib_ref] A method to integrate benchmark dose estimates with genomic data to assess..., Thomas [/bib_ref] Determination of internal dose is important in risk assessment and provides highly relevant information that is more closely associated with disease response than external exposure estimates. [bib_ref] Biomonitoring-based risk assessment for hexabromocyclododecane (HBCD), Aylward [/bib_ref] The capability of -omics technologies to generate information that can be used for internal dose estimation and response markers will be important in their use in risk assessment.DNA mutation or cytogenetic change Susceptibility Inherent or acquired sensitivities or resistance in response to specific exposures Genetic polymorphisms in metabolic activation/deactivation enzymes Epigenetics effects may also have an important role in the development of disease. For example, gene silencing, which is the interruption or suppression of the expression of a gene at transcriptional or translational levels, can occur with hypermethylation of DNA. [bib_ref] DNA methylation in cancer: a gene silencing mechanism and the clinical potential..., Fukushige [/bib_ref] Biomarkers of hypermethylation of DNA may be useful as early cancer detectors and therefore may have utility in risk assessments.
Although biomarkers have been identified using -omics technologies, there is no well-established standardized application of these technologies in using biomarkers in risk assessment [fig_ref] TABLE I: Glossary of Key Terms [/fig_ref]. Involvement of multiple molecular pathways in disease as in systems biology creates complex data analysis/interpretation challenges in validating associations between outcomes and sets of biomarkers.
The concept of the exposome, which encompasses all exposures over a lifetime, has the potential to improve risk assessment. [bib_ref] Complementing the genome with an "exposome": the outstanding challenge of environmental exposure..., Wild [/bib_ref] The exposome will rely on -omics or other high throughput techniques for the identification of biomarkers of exposure and effect. Multiplex profiling (metabolomics, proteomics, and transcriptomics) is now being used along with complementary assays for the most comprehensive and informative views of biological systems. [bib_ref] Use of biomarkers and surrogate endpoints in drug development and regulatory decision..., Lesko [/bib_ref] The exposome has the potential to offer more comprehensive exposure data that can be used to develop more accurate exposure profiles to improve risk assessments.
Most common chronic diseases involve the interaction of multiple exposures and biological pathways that ultimately lead to disease. System biology approaches have been used to study a variety of diseases such as epilepsy and metabolic syndrome and exposures such as particulate matter found in air pollution. [bib_ref] Systems biology of human epilepsy applied to patients with brain tumors, Mittal [/bib_ref] [bib_ref] Metabolic syndrome: from epidemiology to systems biology, Lusis [/bib_ref] [bib_ref] Epigenetic regulation in particulate matter-mediated cardiopulmonary toxicities: a systems biology perspective, Wang [/bib_ref] However, systems biology and how different biological processes may interact with one another to result in disease needs to be better understood to be useful in risk assessment.
The NRCidentified several strategies to use biomarkers of effect to extrapolate dose and evaluate dose response. Physiological-based pharmacokinetic (PBPK) modeling can describe the relationship between external exposure and the internal dose (e.g., blood or tissue concentration of a toxicant) that simulate the toxicity pathways of a chemical. PBPK models can also be used to estimate an external dose (i.e., the relevant real world exposure) that would correspond to the doses used in in vitro and in vivo test systems, as well as in dose-response models to predict the environmental exposure needed to elicit a toxic response.Extrapolating in vitro dose-response data to predict responses in vivo has been a challenge because the doses applied in vitro have typically been much higher than cells in vivo (e.g., in the lungs) would experience even at occupational exposures.(42) An example of one approach is a range of in vitro doses (∼0.2-68 µg/ml) that was proposed based on estimates of the equivalent doses to human lung cells after either 24-hr or a 45-year working lifetime exposure to 1 mg/m 3 of poorly soluble particles. [bib_ref] Informing selection of nanomaterial concentrations for ToxCast in vitro testing based on..., Gangwal [/bib_ref] Uncertainty about how well in vitro studies predict responses in in vivo systems includes the effect of dose rate and the role of other cells and processes in determining the in vivo response. [bib_ref] Nanotoxicology: in vitro-in vivo dosimetry, Oberdörster [/bib_ref] An example of in vitro assays that show predictive trends of in vivo dose-response relationships is for biomarkers
## Table v. examples of -omics technologies
## Technology parameters
Proteomics Involves the identification, characterization and quantitation of expressed proteins in biological samples. Provides complementary functional information to genomics.
## Metabolomics
Studies the metabolic products of the human body and provides a comprehensive view of cellular metabolic changes in small molecules and by-products. [bib_ref] Novel technologies for the discovery and quantitation of biomarkers of toxicity, Collins [/bib_ref] The metabolomics-driven approaches may provide insight into complex biochemical processes and the MOA and toxicity of chemicals. [bib_ref] Metabolomics: a global biochemical approach to drug response and disease, Kaddurah-Daouk [/bib_ref] [bib_ref] Applications of metabolomics in drug discovery and development, Wishart [/bib_ref] Toxicogenomics Brings together toxicology, genetics, and molecular biology such as transcriptomics, proteomics, and environmental health to understand the response of an organism to an external insult. The promise of this technology is that biomarkers of exposure and effect can be elucidated. [bib_ref] Toxicogenomic profiling of chemically exposed humans in risk assessment, Mchale [/bib_ref] Insight into the mechanism of action and low-dose effects are other benefits.
## Table vi. uses of biomarkers in hazard characterization and dose-response analysis
Aids in the identification of mode of action in support of risk assessment Extends the dose-response curve to lower levels of exposure Addresses uncertainty and variability including interspecies differences and identifying susceptible population of inflammation in lung epithelial cell cultures and acute pulmonary inflammation in rats. [bib_ref] Concordance between in vitro and in vivo dosimetry in the proinflammatory effects..., Donaldson [/bib_ref] [bib_ref] Concept of assessing nanoparticle hazards considering nanoparticle dosemetric and chemical/biological response metrics, Rushton [/bib_ref] Establishing a dose of concern is a primary goal of risk assessment. Several approaches that use an internal dose have also been described, including internal dose measures such as biological exposure indicesand biomonitoring equivalents.Additionally, no observed effect levels and benchmark dose estimates can be applied for both internal and external dose measures. [bib_ref] A new method for determining allowable daily intakes, Crump [/bib_ref] [bib_ref] Calculation of benchmark doses from continuous data, Crump [/bib_ref] These dose estimates may be used as points of departure to estimate exposures associated with lower (or presumed no) disease risk. [fig_ref] FIGURE 2: Biologic responses as a result of an exposure [/fig_ref] Genotype-exposure interactions are particularly important for occupational and environmental diseases. Environmental and occupational triggers may interact with genetic factors to initiate the disease process or influence the clinical outcomes including time to onset, severity of the response, or dose. There has been little effort in incorporating genetic information into the risk assessment process, although the advantage of such data in improving accuracy has been discussed. [bib_ref] Hirschhorn: Meta-analysis of genetic association studies supports a contribution of common variants..., Lohmueller [/bib_ref] [bib_ref] A statistical model for assessing genetic susceptibility as a risk factor in..., Demchuk [/bib_ref] [bib_ref] Modification by ALAD of the association between blood lead and blood pressure..., Scinicariello [/bib_ref] Currently, the default approach for addressing interindividual variation in susceptibility for threshold effects is to apply a 10X uncertainty factor. [bib_ref] Human variability and noncancer risk assessment: an analysis of the default uncertainty..., Renwick [/bib_ref] Note that this factor is not intended to cover the entire range of human variability. Instead, this factor addresses the difference between a "safe dose" estimate in the general population and the "safe dose" estimate in the population of interest. [bib_ref] Differential sensitivity of children and adults to chemical toxicity. II. Risk and..., Dourson [/bib_ref] While the default size of the inter-individual factor is 10X, smaller or larger factors may be applied if supported by the available data, resulting in refined estimates of human variability. Criteria for the use of data to support other factors have been developed by the International Programme on Chemical Safety (IPCS).Biomarkers can play a critical role in describing the distribution of responses to a specified dose. This concept is illustrated in [fig_ref] FIGURE 3: Frequency distribution of a biomarker [/fig_ref] , in which the distribution (e.g., the biomarker for a physiologically important response) is shifted in the susceptible populations or life stages, resulting in a bi-or multi-modal overall distribution. The shape of the distribution for a given biomarker would depend on how it is distributed in the population (e.g., whether associated with specific gene alleles or results from multiple causes).
# Methods and approaches
## Risk assessment methods and issues
The goal of human health risk assessment is to predict the likelihood of adverse health effects before they manifest in a population. Different types of studies can provide information that has utility in risk assessments. Epidemiologic studies are important for the assessment of toxic effects directly in humans because no interspecies extrapolation is needed. Such studies are also important in estimating population-based, exposureattributable risks. The most important challenges related to epidemiological studies are the difficulties in precise estimation of exposure, existence of confounding variables such as other exposures and considerable inter-individual variation including genetic make-up, physiological, nutritional, and lifestyle differences. Such studies are also costly and time consuming and have limitations in characterizing dose-response relationships, causal mechanisms, and extrapolating to low doses in risk assessment. [bib_ref] Epidemiology and risk assessment, Samet [/bib_ref] [bib_ref] Methodologic issues in using epidemiologic studies of occupational cohorts for cancer risk..., Stayner [/bib_ref] Extrapolations from animal studies to humans are confounded by a number of issues, including species-specific differences in uptake and response, homogeneity of test animals as compared to heterogeneity of human populations and shortterm testing as compared to complex lifetime exposure, as well as uncertainties due to gaps in the available data. Extrapolation is further complicated by levels and routes of exposure, as these factors can differ greatly between animal models and real-life exposure scenarios. With regard to interspecies extrapolation, several factors must be considered, such as dose normalization for the differences in body size, metabolic rate, variability in toxicokinetics of the chemical, and sensitivity of the target for toxicity. Occupationally exposed populations have considerable physiological and genetic variability in such factors as metabolic capacity and in toxicity response.
Efforts have been undertaken to harmonize dose-response relationships for cancer and non-cancer endpoints [bib_ref] Harmonization of cancer and noncancer risk assessment: proceedings of a consensusbuilding workshop, Bogdanffy [/bib_ref] focusing on MOA as the basis for selecting dose-response models and determining extrapolation approaches. [bib_ref] IPCS framework for analyzing the relevance of a noncancer mode of action..., Boobis [/bib_ref] In general, the default science policy choice based on MOA assumes that a threshold would not exist for substances that interact directly with DNA. This is based on the idea that damage to one DNA molecule could be fixed as a mutation and clonally expand to cancer or result in other effects, such as developmental toxicity. MOAs that do not involve direct DNA reactivity (e.g., cytotoxicity leading to either necrosis or to regenerative cell proliferation and cancer) are generally considered to have biological response thresholds, due to the existence of repair and redundant cellular processes.However, when data are available that provide strong evidence for alternative modes of action, these data may replace default assumptions in risk assessment and OEL derivation. For example, NIOSH [bib_ref] Occupational Exposure to Titanium Dioxide, Department [/bib_ref] used evidence concerning a secondary genotoxic MOA (via persistent inflammation) to inform selection of the nonlinear (but also non-threshold) dose-response models used to estimate the working lifetime risk of lung cancer from inhalation exposure to the poorly-soluble particulate titanium dioxide (TiO 2 ).
There has been considerable discussion in the risk assessment community recently concerning the observation of nonthreshold behavior for chemicals that do not interact with DNA. [bib_ref] State-of-the-science workshop report: issues and approaches in low-dose-response extrapolation for environmental health..., White [/bib_ref] [bib_ref] Linear low-dose extrapolation for noncancer heath effects is the exception, not the..., Rhomberg [/bib_ref] A threshold response for a given agent may be difficult to detect in a population (e.g., a statistical doseresponse model may not be able to exclude zero as a possible [bib_ref] Estimating risk from ambient concentrations of acrolein across the United States, Woodruff [/bib_ref]. Reproduced from Environmental Health Perspectives.
threshold dose), even if the MOA evidence indicates a threshold is plausible. Reasons for observing non-threshold behavior in a population for non-carcinogens include variability in individual threshold responses or exposures that contribute to an existing disease process. [bib_ref] Fundamental carcinogenic processes and their implications for low dose risk assessment, Crump [/bib_ref] [bib_ref] Susceptibility differences in chemical carcinogenesis linearize the dose-response relationship: threshold doses can..., Lutz [/bib_ref] Rhomberg et al. [bib_ref] Linear low-dose extrapolation for noncancer heath effects is the exception, not the..., Rhomberg [/bib_ref] suggested some alternative explanations, such as measurement error at low exposures in epidemiology studies, for not detecting a threshold in human studies when a threshold is observed in animal studies. Additionally, the observation of a threshold may be influenced by factors including variability and sample size in both animal studies and epidemiology studies.
Risk estimates based on extrapolating high-dose animal studies to humans may be particularly sensitive to assumptions about the MOA and shape of dose-response relationships including threshold/non-threshold assumptions. Thus, there may be considerable uncertainty in extrapolation from animal studies when the doses are considerably higher than those relevant to OEL development (e.g., if the MOA that occurs at a high dose is not relevant to that occurring at a much lower dose). Additional uncertainty may occur from temporal extrapolations, which could result in over-or underestimation of the risk of long-term exposure. [bib_ref] Considerations of temporal toxicity challenges current default assumptions, Jarabek [/bib_ref] [bib_ref] Time extrapolation and interspecies extrapolation for locally acting substances in case of..., Kalberlah [/bib_ref] Even when the MOA is known, statistical arguments cannot resolve the uncertainty in low-dose extrapolation, and so science policy choices (e.g., default approach of linear low dose extrapolation for carcinogens in the absence of strong evidence indicating otherwise) are needed in risk assessment.
One of the advantages of using biomarkers of effect is that they can help to reduce the need for extrapolation, allowing instead evaluation of effects in the dose range of interest and in the species of interest (e.g., when human cells are tested in vitro). Under ideal situations, the MOA is used to identify appropriate biomarkers, which are then evaluated sufficiently close to the dose range of interest, so that mathematical curve fitting can be used to more directly estimate risk, rather than relying on the cruder approaches of linear extrapolation (assuming no threshold) or uncertainty factors (assuming a threshold response).
The use of precursor effect data or biomarkers of early effect is gaining increased scrutiny for use in risk assessments. A challenge is that many of these biomarkers lack validation. [bib_ref] Assessing biomarker use in risk assessment-a survey of practitioners, Maier [/bib_ref] The basis for extrapolation between the biomarker and the toxicological outcome needs to be established so that a dose associated with a low risk of an adverse health effect can be estimated.
## Direct dose-response using early effects data
The analysis of -omic dose-response studies has traditionally utilized analysis of variance (ANOVA) approaches together with pair-wise comparisons between dose groups and the corresponding control. [bib_ref] Statistical design and the analysis of gene expression microarray data, Kerr [/bib_ref] The ANOVA identifies genes that are significantly altered as a function of dose while the pair-wise comparisons identify genes that are significantly altered between specific dose pairs. The ANOVA approach for analyzing -omic dose-response studies is analogous to the methods used to define lowest observed adverse effect levels (LOAEL) or no observed adverse effect levels (NOAEL) for other toxicological endpoints [fig_ref] TABLE I: Glossary of Key Terms [/fig_ref]. For applying -omic dose-response data to chemical risk assessment, the traditional ANOVA approach faces several challenges in that dose spacing and the experimental sample size can have a dramatic impact on the final NOAEL and LOAEL, and the approach does not account for variability in the estimate of the dose-response or the slope of the dose-response curve.
To utilize -omic dose-response data within the existing risk assessment paradigm, benchmark dose (BMD) methods have been used to fit a statistical model to the dose-response data and to identify a dose that causes a defined change in the endpoint of interest. [bib_ref] Application of transcriptional benchmark dose values in quantitative cancer and noncancer risk..., Thomas [/bib_ref] [bib_ref] A method to integrate benchmark dose estimates with genomic data to assess..., Thomas [/bib_ref] [bib_ref] BMDExpress: a software tool for the benchmark dose analyses of genomic data, Yang [/bib_ref] The application of the BMD method provides several advantages including better use of dose-response information, more appropriate reflection of experimental sample sizes, and the lack of constraint to experimental doses. [bib_ref] The benchmark dose method-review of available models, and recommendations for application in..., Filipsson [/bib_ref] In this analysis, the dose and individual gene response data
## Effect or no effect level general effect
## Noel
No observed biological effects in the exposed population NOAEL Effects may be seen at this level but not considered to be adverse a) Enzyme induction or other biochemical change, consistent with possible mechanism of action, with no pathological changes and no change in organ weights b) Enzyme induction and subcellular proliferation or other changes in organelles, consistent with possible mechanism of action, but not other apparent effects. c) Hyperplasia, hypertrophy, or atrophy, but no changes in organ weights LOAEL Lowest exposure concentration where adverse effects are seen between the exposed and the control population. a) Reversible cellular changes including cloudy swelling, hydropic change or fatty changes b) Degenerative or necrotic tissues with no apparent decrement in organ function FEL Exposure level in which unmistakable adverse effects are seen that are likely to be irreversible a) Pathological changes with definite organ dysfunctions b) Pronounced pathological changes with severe organ dysfunction with long-term sequelae Notes: NOEL -No Observed Effect Level; NOAEL -No Observed Adverse Effect Level; LOAEL -Lowest Observed Adverse Effect Level; FEL -Frank Effect Level. Adapted from EPA.are fit with the standard set of statistical models used in BMD analysis. A single model is selected for each gene based on fit, modeling complexity, and the BMD and associated lower confidence limit (BMDL).
To allow investigators to interpret the -omic data and provide context for the observed BMD values, public and commercial databases are used to group genes into functional processes and signalling pathways. [bib_ref] KEGG for representation and analysis of molecular networks involving diseases and drugs, Kanehisa [/bib_ref] [bib_ref] Quantitative knowledge-based analysis in compound safety assessment, Bureeva [/bib_ref] The choice of database depends on the context required for interpreting the -omic dose response study. For certain studies, a pathway-based analysis may provide a better understanding of the underlying perturbations in the signaling networks while in other studies, an analysis focused on cellular-processes may provide better linkage with the phenotypic effects of the chemical. The BMD and BMDL values for the individual genes are summarized to represent the general behavior of the process or signaling pathway as a function of dose. In most cases, the mean or median BMD and BMDL are sufficient to capture the general dose-related perturbation of the category or pathway. In certain studies, the transcriptional BMD values for specific cellular biological processes and pathways showed a high degree of correlation with traditional non-cancer and cancer-related apical BMD values. [bib_ref] Application of transcriptional benchmark dose values in quantitative cancer and noncancer risk..., Thomas [/bib_ref] [bib_ref] A comprehensive statistical analysis of predicting in vivo hazard using high-throughput in..., Thomas [/bib_ref] Many of the correlated processes and pathways had been implicated in non-cancer and cancer disease pathogenesis. Subsequent studies have demonstrated a high degree of correlation between transcriptional BMD values for the most sensitive pathway response and traditional non-cancer and cancer-related apical BMD values. [bib_ref] Temporal concordance between apical and transcriptional points of departure for chemical risk..., Thomas [/bib_ref] Early effects data can provide evidence about the MOA and the shape of the dose-response relationship for disease development. Epigenetic effects may alter down-stream responses and outcomes. However, to most effectively use early response and systems biology data in risk assessment and OEL derivation, predictive models are needed to link the early response with the probability of developing the frank effect (conditional on the early effect).
An example MOA involving early responses and frank effect is persistent lung inflammation associated with development of cancer. [bib_ref] Pneumoconiosisrelated lung cancers: preferential occurrence from diffuse interstitial fibrosis-type pneumoconiosis, Katabami [/bib_ref] This effect has also been observed in animals related to inhaled, poorly-soluble particlesincluding TiO 2 . The MOA for rat lung cancer from inhaled poorly soluble particles is generally considered to involve persistent pulmonary inflammation, which causes oxidative DNA damage.Driscoll et al. [bib_ref] Effects of particle exposure and particleelicited inflammatory cells on mutation in rat..., Driscoll [/bib_ref] observed an increased mutation frequency in the hypoxanthine-guanine phosphoribosyl transferase gene (hprt mutations are detrimental lesions caused by oxidative damage to DNA) in alveolar type II cells from rats treated with a high mass dose (100 mg/kg) of fine-sized TiO 2 or other types of poorly-soluble particles. In vitro, hprt mutation frequency was also increased in an alveolar epithelial cell line (RLE-6TN) following co-incubation with inflammatory cells (alveolar macrophages and neutrophils) derived from bronchoalveolar lavage fluid from particle-treated rats. [bib_ref] Effects of particle exposure and particleelicited inflammatory cells on mutation in rat..., Driscoll [/bib_ref] Addition of catalase (an enzyme which protects cells against oxidative damage) to these co-incubations inhibited the increase in hprt mutations. These studies support a role of inflammatory cellderived oxidants in particle-associated mutagenesis.
In risk assessment and development of recommended exposure limits for fine and ultrafine TiO 2 , NIOSH [bib_ref] Occupational Exposure to Titanium Dioxide, Department [/bib_ref] used statistical models of animal dose-response data for lung cancer and pulmonary inflammation to estimate the working lifetime risks. On the basis of a secondary genotoxic mechanism, prevention of persistent lung inflammation would be expected to prevent lung cancer by that mechanism. However, evaluation of the rat subchronic inflammation data did not show evidence of a threshold (although the dose-response relationship was nonlinear). Rat-and human-based excess risk estimates for lung cancer from working lifetime exposures to inhaled poorly-soluble particles were compared. The particles evaluated include those for which long-term dose-response data are available in both species, i.e., coal dust, carbon black, titanium dioxide, silica, and diesel exhaust particulate. The excess risk estimates derived from the rat data were generally lower than those derived from the human studies, and none of the ratand human-based risk estimates were significantly different (all p-values>0.05). [bib_ref] Stayner: Rat-and human-based risk estimates of lung cancer from occupational exposure to..., Kuempel [/bib_ref] Given the limited data available to quantitatively evaluate the relationship between inflammation and lung cancer in rats or humans, NIOSH derived the Recommended Exposure Limits on the basis of rat dose-response data for lung tumors. NIOSH estimated the human-equivalent, 8-hr time-weighted average concentrations associated with <1/1000 excess risk of lung cancer over a working lifetime, derived from the nonlinear dose-response models fit to the rat data. [bib_ref] Occupational Exposure to Titanium Dioxide, Department [/bib_ref]
## Biologically based dose response (bbdr) models
Risk estimates that rely on default assumptions may be uncertain to the extent that the true relationships differ from those assumptions. This uncertainty arises from the limited data that are available to inform the selection of the doseresponse models and the assumptions used in interspecies and low-dose extrapolations. Risk estimates on the basis of default assumptions may overestimate the risk for a population because the default approaches are intended to be conservative in the absence of chemical-specific data. [bib_ref] The use of biologically based modeling in risk assessment, Conolly [/bib_ref] They may also underestimate risk in other cases (e.g., if greater individual variability exists than accounted for in the default assumptions). [bib_ref] Distributions of individual susceptibility among humans for toxic effects. How much protection..., Hattis [/bib_ref] [bib_ref] Human interindividual variability-a major source of uncertainty in assessing risks for noncancer..., Hattis [/bib_ref] By utilizing measurements of biological pathway perturbations, uncertainties in the target tissue dose across species and the influence of exposure routes may be decreased, resulting in more reliable risk assessments. [bib_ref] The use of biologically based modeling in risk assessment, Conolly [/bib_ref] An advantage of a BBDR model is that, by describing key steps in the development of toxic effects, alternative mechanisms of action can be evaluated and compared to the data, to test hypotheses and evaluate the importance of specific assumptions. BBDR models also have the advantage of directly predicting the response at doses of interest, avoiding the threshold/non-threshold dichotomy, but they may require assumptions about the connections between dose and key events. These models can also incorporate inter-individual susceptibility and confounders such as existing diseases and background exposures. [bib_ref] State-of-the-science workshop report: issues and approaches in low-dose-response extrapolation for environmental health..., White [/bib_ref] Although BBDR models have a number of advantages, a key issue in their use is the uncertainties associated with the parameters used in the model, as well as the substantial sensitivity of the model results to the assumptions regarding the underlying mathematical form for intermediate steps in the mechanism of action. [bib_ref] What role for biologically based dose: response models in estimating low-dose risk?, Crump [/bib_ref] However, identification of biomarkers corresponding to these intermediate steps would provide an opportunity to directly address these assumptions and reduce the uncertainty of key parameters. Verification of BBDR model predictions, as well as incorporation of population-based distributions of parameter values, may be needed for wider acceptance of these models in risk assessment and development of OELs. The International Programme on Chemical Safetyguidance on use of physiologically based pharmacokinetic (PBPK) models in risk assessment provides a template to facilitate understanding of models by risk assessors; a key consideration is comparing the uncertainties of the PBPK/BBDR model with those of the default approach.
Biologically informed empirical dose-response modeling provides a bridge between strictly empirical models and full BBDRs. Such approaches are analogous perhaps to compartmental pharmacokinetic models, but can incorporate pharmacodynamic data using biomarkers. Like the compartmental pharmacokinetic models, the biologically-informed empirical dose-response models incorporate some chemical-specific data, but include empirical curve-fitting. The goal of such analytical methods is to improve the qualitative and quantitative description of the biological processes determining the shape of the dose-response curve, without investing the resources needed to develop and verify a BBDR model. An advantage of these approaches is the use of quantitative data on early events (biomarkers) to extend the overall dose-response curve to lower doses using biology, rather than being limited to the default choices of linear extrapolation or uncertainty factors. Using biomarkers to extend the dose-response curve towards the dose region of interest also offers the potential for better description of the dose-response relationship of chemicals with a MOA that includes contributions from both DNA-reactive and non-DNA reactive components.
Allen et al.developed such a model as a proof of concept for predicting risk of lung cancer given persistent lung inflammation from chronic inhalation of TiO 2 in rats. A series of cause and effect functions, fit using a likelihood estimation approach, were utilized to describe the relationships between successive key events leading to the ultimate tumor response. This approach was used to evaluate a hypothesized pathway for progression from a biomarker of exposure (lung burden), through several intermediate potential biomarkers of effect, to the clinical effect of interest (lung tumor production).
Another approach to biologically informed empirical doseresponse modeling was demonstrated by Hack et al., [bib_ref] A Bayesian network model for biomarker-based dose response, Hack [/bib_ref] who used a Bayesian network model to integrate exposure biomarkers to conduct an exposure-dose-response assessment for acute myeloid leukemia resulting from exposure to benzene. The network approach was used to evaluate and compare individual biomarkers and quantitatively link the biomarkers along the exposure-disease continuum. This work provides a quantitative approach for linking changes in biomarkers of effect both to exposure information and to changes in disease response. Such linkage can provide a scientifically valid point of departure that incorporates precursor dose-response information without being dependent on the difficult issue of a definition of adversity for precursors.
More classical mathematical approaches also have the potential for linking biomarkers to adverse effects. For example, the Hill model describes the biology of a chemical binding to a receptor, a key event in many receptor-mediated MOAs. Budinsky et al. [bib_ref] Human and rat primary hepatocyte CYP1A1 and 1A2, Budinsky [/bib_ref] used the Hill model to compare the doseresponse for aryl hydrocarbon receptor-mediated CYP1A1 and CYP1A2 messenger RNA induction and enzyme activity in rat and human hepatocytes exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin, 2,3,4,7,8-pentachlorodibenzofuran, or 2,3,7,8-tetrachlorodibenzofuran. In an extended analysis of genome-wide transcriptomic data from the same experiment, BMD analysis of the gene expression changes showed an average 18-fold cross-species difference in potency among differentially expressed orthologs and similar differences were observed for signaling pathways. [bib_ref] Cross-species comparisons of transcriptomic alterations in human and rat primary hepatocytes exposed..., Black [/bib_ref] The data were used to support the conclusion that humans are less sensitive than rats to these aryl hydrocarbon receptor-dependent end points and to support the use of a modified uncertainty factor for extrapolating between rats and humans.
More general approaches to empirical dose-response modeling are incorporated in standard modeling methods where the mathematical form used for empirical curve-fitting is based on the presumed shape of the biological response. Thus, for example, probit modeling is typically used for modeling lethality data. Similarly, a multistage model has been used for tumor modeling, based on the multi-stage model for cancer. In an example of modifying the standard choice based on biology, Dourson et al. [bib_ref] Evidence-based doseresponse assessment for thyroid tumorigenesis from acrylamide, Dourson [/bib_ref] used the probit model to describe the dose-response for thyroid tumors in rats orally exposed to acrylamide. This choice was based on both improved empirical model fit compared to the multistage model, and the observation that the shape of the probit model better reflected (compared with the default linear extrapolation approach) the mixed MOA of DNA reactivity at low doses and growth stimulation at the higher doses tested in the animal bioassay.
# Conclusions
# Advantages and limitations
Several key advantages to the use of biomarkers in risk assessments exist. Biomarkers are used to identify the MOA and can support the MOA in risk assessments rather than relying on general default approaches. Additionally, biomarkers can be used to characterize inter-individual variability by helping to ensure that sensitive populations are identified and adequately addressed in the assessments and to reduce uncertainty in the extrapolation of animal data to humans. [bib_ref] Modification by ALAD of the association between blood lead and blood pressure..., Scinicariello [/bib_ref] [bib_ref] SAR/QSAR methods in public health practice, Demchuk [/bib_ref] Another advantage of biomarkers is hypotheses are tested at doses relevant to human exposures. One ultimate goal of the use of biomarkers is to extend the dose-response curve to the range (or near the range) of the exposures of interest. This would allow one to use the biomarker data more directly to evaluate doseresponse, without having to go to default approaches of linear or nonlinear extrapolation. Such data could be used to establish more appropriate OELs to protect individuals who are at high risk. Systems biology and MOA approaches will also lead to new hypotheses and ways of thinking about chemical risk assessments and hence move the entire field of risk assessment forward.
While early biomarkers of effect have great promise, many limitations and challenges need to be overcome before early effect biomarkers can be reliably used. The whole field of computational toxicology and systems biology is still evolving and results have not been validated in human populations.
Appropriate interpretation and validation of biomarker results is lacking.
## Special issues in applying these approaches for oel setting
Developing OELs on the basis of early effects doseresponse data means that more sensitive, relevant endpoints could be targeted for prevention. If these biomarkers can be validated to ensure they represent an adverse effect, it may be possible to reverse a deleterious exposure before the disease has progressed. These precursor events (i.e., detected using a biomarker) might be preclinical but could be associated with an increased susceptibility to develop the disease effect. Setting OELs to prevent early adverse effects may help to prevent material impairment of health and functional capacity as a result of workplace exposure. However, a challenge is to determine the linkage between early effects, which may not yet constitute material impairment of health and functional capacity, and the later adverse outcomes.
Since the risk of preclinical responses have not been well defined with respect to what those biomarkers mean to health, this presents a challenge in how to utilize early effects data in a standardized, harmonized risk assessment strategy across agents and cancer and non-cancer endpoints, as recommended by the NRC.The use of BBDR models to quantitatively link early preclinical changes to apical endpoints of regulatory concern may mitigate this problem in the future.
Standardization is an important issue in the use of biomarkers, although the issue is not unique to the biomarker-based risk assessments. In an approach based on the NOAEL/LOAEL with uncertainty factors, the NOAEL may be based on a range of different responses or severity of response at the corresponding LOAEL. This severity of the endpoint may be addressed in the magnitude of the uncertainty factor applied to the LOAEL, but this is a relatively crude approach. One of the advantages of the BMDL is that it is based on a response level, but differences in severity of the endpoint can still lead to inconsistencies.
Early biological effects using a systems biology approach and computational toxicology efforts offer great promise for the future of risk assessment. Information on these effects can be generated using HTS providing needed information quicker and cheaper than conventional animal testing. Proof of concept studies in computational toxicology provide early evidence of their promise in utilizing early biomarkers in establishment of dose. [bib_ref] Application of transcriptional benchmark dose values in quantitative cancer and noncancer risk..., Thomas [/bib_ref] [bib_ref] A method to integrate benchmark dose estimates with genomic data to assess..., Thomas [/bib_ref] However, challenges such as standardization and validation still need to be overcome before these methods are used in routine risk assessments.
# Acknowledgments
T he authors would like to acknowledge Dr. Andrew Maier for his contribution and thoughtful insight in the areas of systems biology and risk assessment.
[fig] FIGURE 2: Biologic responses as a result of an exposure. The intersection results in perturbation of biologic pathways. When perturbations are sufficiently large or when the host is unable to adapt because of underlying nutritional, genetic, disease, or life-stage status, biologic function is compromised, and this leads to toxicity and disease.(94) © Elsevier. Reproduced by permission of Elsvier. Permission to reuse must be obtained from the rightsholder. [/fig]
[fig] FIGURE 3: Frequency distribution of a biomarker (physiological parameter) in two hypothetical populations to illustrate the effect of exposure and susceptibility factors. Adapted from Woodruff et al. [/fig]
[table] TABLE I: Glossary of Key Terms [/table]
[table] TABLE VII: Effect Levels, by Severity, That are Considered in the Derivation of Exposure Limits [/table]
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Migration of Alpine Slavs and machine learning: Space-time pattern mining of an archaeological data set
The rapid expansion of the Slavic speakers in the second half of the first millennium CE remains a controversial topic in archaeology, and academic passions on the issue have long run high. Currently, there are three main hypotheses for this expansion. The aim of this paper was to test the so-called "hybrid hypothesis," which states that the movement of people, cultural diffusion and language diffusion all occurred simultaneously. For this purpose, we examined an archaeological Deep Data set with a machine learning method termed time series clustering and with emerging hot spot analysis. The latter required two archaeologyspecific modifications: The archaeological trend map and the multiscale emerging hot spot analysis. As a result, we were able to detect two migrations in the Eastern Alps between c. 500 and c. 700 CE. Based on the convergence of evidence from archaeology, linguistics, and population genetics, we have identified the migrants as Alpine Slavs, i.e., people who spoke Slavic and shared specific common ancestry.
# Introduction
On Easter Monday, 2 April 568 CE, Alboin, son of Audoin and king of the Lombards, led his people on an arduous journey. They were to "abandon the barren fields. . . and come and take possession of Italy." Because from the "west and north is shut in by the range of Alps, they reached it from the eastern side by which it is joined to Pannonia" (Paul the Deacon, Book II, Ch. V. and IX.;.
Historiography records Alboin's success: He founded the Kingdom of Italy the next year and his successors ruled there for almost two centuries, e.g., [bib_ref] Maribor: Obzorja, Bradač [/bib_ref]. However, historiography is less clear about events in the lands that Alboin and his people left behind. These lands include the Eastern Alps; we know little more than that this region was sparsely populated by Romans a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 confidence level for chronology and location) and paradata (e.g., sources for dating). Furthermore, it was expanded to include Late Antiquity sites (400 to 600 CE in our study).
However, this data enrichment focused on a geographically limited subset of data. This subset of 1,105 archaeological sites constitutes the study area of this article. It includes present-day Slovenia, southern Austria (Carinthia, Styria, East Tyrol, parts of Salzburg and Upper Austria) and a small part of northern Italy (the Trieste region) [fig_ref] Fig 1: Map of the study area [/fig_ref].
## Plos one
The data are described in more detail in the data paper [bib_ref] Deep Data Example: Zbiva, Early Medieval Data Set for the Eastern Alps, Š Tular [/bib_ref] and are openly accessible via the Zenodo repository [bib_ref] Early Medieval Data Set for the Eastern Alps. Data sub-set, Š Tular [/bib_ref].
Compared to the entirety of Slavic territories ours is a small study region. But this region is an excellent (if not pivotal) case study for understanding the general processes of the spread of Slavic speakers in Early Middle Ages for three reasons. (i) Archaeologically, this is the only region where data is readily available for advanced spatial analysis, including machine learning (see above). (ii) The historiographical sources are second to none and include the oldest permanent Slavic political entity (Carantania, after 650 CE; e.g.,, the oldest Slavic text other than the canonical Old Church Slavic (ninth-century Monumenta Frisingensia;, and the oldest mention of a member of a specifically Slavic social elite, a župan (iopan Physso, 777 CE. (iii) Linguistically, the area is on the southwestern periphery of the spread of Slavic, bordering Germanic and Romance languages; this is important because peripheries typically preserve archaisms better than centres.
## Space-time cube
Artificial Intelligence (AI) is becoming an ever more integral part of the digital humanities. Here, we focus on machine learning, which is a subset of AI. It refers to a set of data-driven algorithms and techniques that automate data prediction, classification, and clustering. Machine learning is rapidly being adopted by archaeologists interested in the analysis of geospatial data, material culture, texts, and artistic data [bib_ref] Machine Learning Arrives in Archaeology, Bickler [/bib_ref] [bib_ref] Machine Learning for Cultural Heritage: A Survey, Fiorucci [/bib_ref] and is most often used in combination with Big Data. Among the most prolific fields for machine learning within archaeology is airborne LiDAR data, e.g. overview in [bib_ref] Documentation of Archaeology-Specific Workflow for Airborne LiDAR Data Processing, Lozić [/bib_ref].
Currently, most machine learning processing takes place in the Phyton and R programming environments. This means that it requires coding and is therefore not readily accessible to most archaeologists. In this paper, we have used the only off-the-shelf pipeline that can be readily applied to archaeology: The toolset for space-time pattern mining in ArcGIS Pro v. 2.9 (ESRI, Redlands CA, USA). A similar pipeline has already been demonstrated on archaeological data [bib_ref] Temporal Analysis Method for Archaeological Sites, Cui [/bib_ref] , but to our knowledge ours is the first time this method was used to test a specific archaeological hypothesis.
Some comments on terminology are in order. "Space Time Pattern Mining" is a commercial name for the toolset used in this work [bib_ref] How Create Space Time Cube works, Esri [/bib_ref]. We classify our method as machine learning, following [bib_ref] Machine Learning for Cultural Heritage: A Survey, Fiorucci [/bib_ref]. They list the methods used in the cultural heritage domain, which are similar to the time series clustering, under unsupervised machine learning. In addition, machine learning is used in the background of this software to enable, for example, intelligent data-driven defaults.
Why use space-time pattern mining? Whenever archaeologists (or anyone, for that matter) look at a map, we inherently begin to turn that map into information by finding patterns and assessing trends. However, sometimes the patterns in the data are too complex to be clustered, observed and aggregated by the human eye. In such cases, we can use space-time pattern mining tools to answer our questions confidently, objectively, and repeatably.
The first step in space-time pattern mining is to organize the data. In this case, the data is incorporated into what is known as the "space-time cube model." This model is based on Torsten Hägerstand's time geography, which introduces the time axis into the traditional Cartesian coordinate system, e.g., [bib_ref] Geovisualization and time: new opportunities for the space-time cube, Kraak [/bib_ref]. It can be thought of as a three-dimensional cube consisting of space-time fields, where the dimensions x and y represent space and the dimension z represents time [fig_ref] Fig 2: Graphical representations of selected methods [/fig_ref]. The data is stored in the so-called "space-time NetCDF cube." The process of constructing the space-time cube data model is complex and beyond the scope of this article, but it is well documented, e.g., [bib_ref] Temporal Analysis Method for Archaeological Sites, Cui [/bib_ref].
The concept of the space-time cube is familiar to archaeologists. At its core, it is an application of the way we perceive (or have perceived in the past) archaeological excavations: In spatial quadrants and time phases. The quadrants in the xy grid are constant throughout the excavation, and the phases stack on top of each other, with the earliest at the bottom and the latest at the top.
For our case study, we aggregated data into 5 km big (y dimension) and 25 years long (z dimension) hexagonal bins. The size of the hexagon was chosen as the largest in which the relevant processes can be observed; in Early Medieval archaeology, it roughly corresponds to the site catchment area of a single settlement, e.g.,. The time interval of 25 years was determined on the basis of the data properties. The time sensitivity of relevant archaeological dating is about half a century, i.e., ± 25 years. However, start and end dates can often be a quarter of a century, rarely as brief as decades or even years. The accuracy of our dates is therefore 50 years, but the precision is approximately 25 years. Accordingly, 25-year intervals were chosen for analysis, but the accuracy of the data requires that archaeological interpretation be limited to 50-year intervals.
This method is very sensitive to the difference between no data (areas not analyzed) and null data (areas analyzed, but not found to contain any known sites). To account for this, we limited the space-time cube to the area used for data collection. Additionally, we excluded areas higher than 1,400 m above sea level [fig_ref] Fig 1: Map of the study area [/fig_ref] shades of brown). In our region, this altitude delineates the highest valley settlements from the lowest high-mountain pastures. The latter were excluded from the analysis because they are specialized seasonal settlements that were always dependent on valley settlements.
## Time series clustering
Clustering is one of the most widely used machine learning techniques in the field of cultural heritage [bib_ref] Machine Learning for Cultural Heritage: A Survey, Fiorucci [/bib_ref]. Its goal is to organize similar data into homogeneous groups or clusters. Clusters are formed by grouping objects that have maximum similarity with other objects within the cluster and minimum similarity with objects in other clusters. For large and complex data sets, unsupervised approaches offer the best solution. Time series clustering is a type of unsupervised clustering used for data with a temporal component [bib_ref] TSclust: An R package for time series clustering, Montero [/bib_ref] [bib_ref] Time-series clustering-A decade review, Aghabozorgi [/bib_ref].
The concept of time-series clustering is deeply familiar to archaeologists having been used since the nineteenth century. At that time, for example, the three-age system, which divides the development of human civilization into the Stone Age, Bronze Age, and Iron Age, was defined by clustering similarly dated stone/bronze/iron artefacts. As most archaeologists know from experience, such clustering is relatively easy for a few artefacts or sites but becomes daunting when the numbers run into the hundreds or thousands of objects or sites. In such cases, unsupervised time series clustering can be used.
In this article, we applied time series clustering to classify sites into chronological groups. In each group, the chronology (start date, end date) of the sites is more similar to each other than that of the sites outside the group.
The similarity between the clusters is measured by the so-called "pseudo-F statistic." The larger the pseudo-F value, the more different each cluster is from the other clusters. There are several ways to calculate the pseudo-F statistic, each depending on which characteristics of the time series are considered important. In our experience, the most appropriate for archaeology is the "Profile (Fourier)" method, i.e., method based on Fourier series periodic function. It is used to cluster time series that have similar, smooth, and periodic patterns over time [bib_ref] How Create Space Time Cube works, Esri [/bib_ref].
This method lends itself to the analysis of archaeological processes because they usually follow a consistent pattern: A gradually introduced innovation is followed by a peak of use and a steady decline. Thus, archaeological processes can be compared to seasons, where temperature follows a consistent annual pattern, with higher temperatures in summer and lower temperatures in winter. The "Profile (Fourier)" method is best suited to finding locations that have the most similar annual temperature patterns, for example, to distinguish between locations with mild and severe winters. A season in this example represents an archaeological phase or period.
In our case, we opted to ignore the range, i.e., the magnitude of the values in each period. To extend the analogy above, ignoring the range causes the change of seasons in two places occurring at the same times to be considered similar, even though the actual temperatures are different.
## Modified emerging hot spot analysis
Spatial analysis is often called upon to determine the density of observed phenomena, and one of the most common tools to do this in archaeology is the so-called "hot spot analysis." It uses the Getis-Ord G � statistic to calculate z-scores and p-values within a given spatial neighborhood. These indicate whether the observed spatial clustering of high and low values is more (hot spot) or less (cold spot) pronounced than would be expected from a random distribution, e.g.,.
In this article, we have used an emerging hot spot analysis that examines the clustering of high and low values over time, in addition to spatial trends. The space-time cube is evaluated bin-by-bin, and each bin is analyzed relative to its space-time neighbors. Thus, each site is related not only spatially but also temporally to neighboring sites. The result is similar to the traditional hot spot analysis, except that it is in 3D (where the z dimension represents time).
Such a result can deliver an overwhelming amount of information. Therefore, the tool evaluates the trends of hot spots and cold spots over time using the Mann-Kendall trend test, e.g., [bib_ref] A modified Mann-Kendall trend test for autocorrelated data, Hamed [/bib_ref] and categorizes each location in the study area accordingly. For example, a location is considered a consecutive hot spot if it has an uninterrupted series of statistically significant hot spot bins over the latest time step intervals, but less than 90% of the total [bib_ref] How Create Space Time Cube works, Esri [/bib_ref]. The resulting 2D representation of trends can be termed a "trend map." However, the trend map provided by the tool is not suitable for archaeology for two reasons. First, it assumes that the latest records are the focus of analysis. Second, it was designed for data sets much larger than ours and those of most archaeological studies.
We therefore modified the trend map by focusing on chronological periods previously calculated by the time series clustering method. For example, a location was considered a first period consecutive hot spot if it had an uninterrupted hot spot series of at least 100 years within the first period (detailed description in S1 . We term this an "archaeological trend map".
In emerging hot spot analysis, the spatial and temporal neighborhoods have a significant influence on the results. We found, through empirical observation, that the best results for hot spots and cold spots were obtained with different settings. For cold spots: fixed distance method with 20 km neighborhood, and time step three. For hot spots: k-nearest neighbors (kNN) method with six spatial neighbors, time step one [bib_ref] How Create Space Time Cube works, Esri [/bib_ref].
Therefore, we have introduced another archaeology-specific modification to the tool. For the purposes of this article, we superimposed the cold spots derived using the first settings with the hot spots derived using the second settings in a single visualization. We refer to this method as "multiscale emerging hot spot analysis."
To ensure the highest level of methodological transparency, reproducibility, and transparency and to reduce the time researchers spend replicating the work of other research groups, we provide the ready-to-re-use data in GIS format and the GIS protocol (S1 Appendix).
## Theory
## Consilience
The specifics of archaeological inference, e.g.,, are not often outlined in articles such as ours. In this case, however, it is necessary because academic passions on the question of the migration of the Slavs have long been running high, and the methods of inference are often scrutinized. Moreover, this topic is invariably interdisciplinary, but the interdisciplinarity is achieved through a variety of approaches.
In order to enrich this discussion with the most objective archaeological information possible, we have chosen to base our inference on consilience of induction. Consilience, also known as "convergence of evidence," is a scientific principle that states that the same conclusion is much stronger when drawn from independent and unrelated sources. Confidence is strongest when evidence from different fields is considered because the methods and/or data are different.
Although it is rarely referred to by its name, this principle is popular in archaeology. For example, consilience is applied whenever radiocarbon dating is invoked to support archaeological dating.
It is important to distinguish between consilience, where conclusions are drawn independently before being correlated, and the more common interdisciplinary approach to the study of Slavic migrations, where data from different fields is compiled to draw a unified conclusion with a mix and match approach [fig_ref] Fig 2: Graphical representations of selected methods [/fig_ref]. To this end, we have been careful to consider only information from each field that has not been influenced by findings from another field. For example, in the Discussion we consider linguistic information, but disregard the conclusions drawn on the same subject matter using supporting evidence from archaeology. We also take care to include only interpretations reached by domain specialists, as reinterpretations by non-specialists can be problematic.
## Material culture as ethnicity, identity, and habitus?
The main archaeological argument for the migrations of the Slavs is based on the association of the Slavs with various archaeological cultures or habitus. For instance, the archaeological assemblages of the so-called Prague Culture are associated with the Early Slavs, e.g., [bib_ref] Od Bachórza Do Swiatowida Ze Zbrucza: Tworzenie Się Słowianskiej Europy W Ujęciu..., Biermann [/bib_ref] [bib_ref] Runes from Lány (Czech Republic)-The oldest inscription among Slavs. A new standard..., Macháček [/bib_ref].
From a modern theoretical perspective, this argument draws on Pierre Bourdieu'snotion of habitus; its basic premise is that practical knowledge is embodied in daily practices and that material culture, including pottery, expresses these practices, e.g.,. However, equating a habitus (e.g., the archaeological assemblages of Prague culture) with a people/tribe/ ethnicity (e.g., the Early Slavs) is an additional step that Bourdieu did not anticipate. In much of the literature after the mid-1960s, the notion that material culture is more or less directly related to cognition of peoples was questioned by many; the acceptance that archaeological cultures simply cannot be directly correlated with ethnicity took hold, e.g., [bib_ref] Ethnische Identitäten als Konstrukte der frü hgeschichtlichen Archä ologie. Germania: Anzeiger der..., Brather [/bib_ref].
Rather than engage in this discourse, we based our argument only on the categories of archaeological data that are indisputable: Location and chronology of the site. Our inference thus eclipses theoretical issues about the associations of material culture with ethnicity, e.g., [bib_ref] Approaches to the Archaeology of Ethnogenesis: Past and Emergent Perspectives, Hu [/bib_ref] , identity, e.g.,, or habitus, e.g.,. Whether or not the Prague Culture archaeological assemblages are associated with the Early Slavs was immaterial to our conclusions.
# Results
## Archaeological periodization
The result of our time series clustering is the archaeological periodization of sites. The most archaeologically meaningful result is when the data is clustered into three periods (pseudo-F statistic value 421.595). These are the Late Antiquity and two Early Middle Ages periods [fig_ref] Fig 3: Archaeological periodization with time series clustering [/fig_ref]. The latter two correspond to the traditional periodization of the jewelry into the Carantanian and the Köttlach phases, e.g.,or groups A/B and C of Eichert. Three important conclusions may be drawn from these results. The first is, that the general increase in sites between 400 and 500 CE and the decrease after 1000 CE does not reflect reality, as we know it from numerous sources, e.g.,. Rather, it reveals the weakness of the underlying data set: The period before 500 CE has not been collated systematically, and data for the period after 1000 CE (which, until recently, was not considered relevant to archaeology in the region, e.g.,is lacking. Regardless, the present data set is suitable for the study of the half millennium between 500 and 1000 CE, which was the aim.
Second, unlike changes in material culture, changes in landscape are more gradual and often overlap. For example, the time series of Late Antiquity does not end until 1000 CE, as some sites exhibit continuity from Late Antiquity onward (for example, the town of Kranj, e.g.,. The results of time series analysis in archaeology are therefore complex and must be interpreted with great care.
Third, we substantiated the long-established periodization of the Early Middle Ages into two periods by an independent source of data: The chronology of sites rather than the typology of jewelry. This, then, is the first quantitative evidence that changes in jewelry styles taking place in the second half of the 9th century were reflected in changes in the archaeological landscape. The most likely explanation is that both changes had the same underlying cause, which however is beyond the scope of this article.
## Archaeological landscape
The emerging hotspot analysis revealed an astonishing quantity and quality of information [fig_ref] Fig 4: Archaeological trend map of the modified categorization of the multiscale emerging hot... [/fig_ref]. Most relevant to our topic are the extensive areas of cold spots in the northern part of the region and the general patchiness, i.e. activity is concentrated in enclaves. In this respect, the archaeological landscape between 500 and 1000 CE differs from both the preceding Roman period settlement and subsequent High Medieval period, which both exhibit a more regular pattern of settlement. This is important in providing a context for understanding various historical processes. For example, the reason it is so difficult for historiographers to define the exact borders of Carniola, e.g.,and Carantania, e.g.,is that in the patchy landscape precise fixed borders most likely never existed.
The main focus of this article was migration. The two tools for detecting migrations in our data were provided by Curta. First, migration must have occurred if settlements and cemeteries suddenly appear in a previously sparsely populated area, i.e. cold spots are immediately followed by hot spots. Second, migration can also be detected by the sudden appearance of a material culture without local traditions or parallels in a given area.
With the first tool, migration was documented in the easternmost part of the study area. In the period between 450 and 500 CE this is a cold spot area, but after c. 500 CE hot spots appear along the river Mura (Ger. Mur). After a period of consolidation until c. 600 CE, a series of small-scale neighbourhood migrations upstream of the Mura and the adjacent Drava (Ger. Drau) rivers is documented by numerous hot spots [fig_ref] Fig 2: Graphical representations of selected methods [/fig_ref].
With the second tool, a migration upstream of the Sava river after c. 675 CE was documented. Between c. 600 and 675 CE, there was a gradual decline in hot spots along the Sava, but between c. 675 and 750 CE there was a reversal of that trend [fig_ref] Fig 2: Graphical representations of selected methods [/fig_ref]. This trend reversal alone could be explained by other causes than migration. The evidence for migration was provided by the time series clustering, which showed a sudden and complete shift in material culture: the number of Late Antiquity sites diminishes dramatically and at the same time Early Medieval sites start appearing [fig_ref] Fig 3: Archaeological periodization with time series clustering [/fig_ref]. This shift has long been known in archaeology as the transition from fortified hilltop settlements to unfortified lowland settlements, e.g.,, which determines the transition from Late Antiquity to the Early Middle Ages.
On the basis of this data, a comment can be made on the size of the migrations. Overall, hot spots interpreted as resulting from the first migration account for only 4% of all hot spots in c. 500 CE, which indicates a relatively small founder population. However, by c. 700 CE, 59% of hot spots can be interpreted as resulting directly or indirectly from both migrations. Although this is a very rough estimate, far from giving a direct indication of the actual number of people involved, it is the best available and by far the most tangible to date, cf., [bib_ref] Slowenien und die Nachbarländer zwischen Antike und karolingischer Epoche. Anfä nge der..., Szameit [/bib_ref]. As such, it is an invaluable foundation for explaining acculturation processes following migrations, which, however, are not the subject of this article.
# Discussion
## Archaeology
Our data thus testifies to two migrations: The first upstream of the Mura and Drava rivers after c. 500 CE, and the second before c. 700 CE upstream of the Sava river. This is an important discovery, but it does not shed light on who the migrants were. Based on the archaeologicaland historiographicalcontext, we can hypothesize that they were Early Slavs. But this hypothesis inherits all the weaknesses of the existing ones, which are based on the controversial presence of archaeological assemblages of Prague Culture and scant written sources.
The hypothesis may, however, be considered the null hypothesis that can be tested with the consilience principle. The new archaeological evidence for two separate migrations allows us to correlate it with interpretations from the linguistics and genetics of modern populations. These two fields of science use completely different data sources and methods than archaeology and have recently made significant advances in understanding Slavic migrations.
## Linguistics
Let us first take a look at linguistics. While a language or dialect may be tied to any number of identities within a given period, a shared linguistic innovation requires a linguistic community, for which the term "founder population" has been proposed.
Modern Slovenian, which is spoken today in the southern part of the research area, belongs to the South Slavic clade, according to the traditional classification of the Slavic languages. There are, however, considerable linguistic similarities between the Slovenian and the West Slavic lects. These similarities were explained either by the existence of a specific link between Slovenian and West Slavic or by a mixed South and West Slavic origin of Slovenian [bib_ref] Ocherk sravnitel'noj grammatiki slavyanskikh yazykov, Bernstein [/bib_ref] [bib_ref] O drevnikh slovensko-zapadnoslavyanskikh svyazyakh. In: Russkoe i slavyanskoe yazykoznanie. K 70-letiyu chl.-korr, Stieber [/bib_ref] [bib_ref] Znachenieto na gramaticheskite, slovoobrazovatelni i leksikalni danni za klasi-fikatsiyata na slavyanskite ezitsi..., Lekov [/bib_ref].
Specific ties between Slovenian and South Slavic on the one hand and West Slavic on the other have recently been demonstrated with a series of phylogenetic NeighborNet networks. The analysis concluded that Slovenian seems to be almost equally close to the West and South Slavic, but distant from the East Slavic, "thus supporting the putative mixed nature of Modern Slovenian" [bib_ref] Genetic Heritage of the Balto-Slavic Speaking Populations: A Synthesis of Autosomal, Mitochondrial..., Kushniarevich [/bib_ref]. It is the latter interpretation that interests us.
In conclusion of the above cited analysis further studies of Slovenian dialects are proposed in order to clarify the position of Slovenian among the Slavic languages. One of such study examined the diatopic distribution and semantic development of � glčěti as the primary neutral verb meaning 'to speak'. It was carried from an emergent dialect of Slavic and is now widespread in present-day central Russia, central Bulgaria, and in Slovenia along the Mura and Drava rivers. Of interest is the hypothesis of possible relationships between the "early Slavic speakers who spoke dialects in which � glčěti played a central role as a verb of speech" and those who did not within modern Slovenia, i.e. the southern part of our study area. The hypothesis states that this dichotomy, together with the -ny-|| -nǫ-isogloss, "can be viewed as inherited pre-migration cleavages", that is, "the dialects of Slavic brought to the subalpine area. . . differed (amongst themselves)"; translated from the Slovenian by B.Š.; the subalpine area mentioned corresponds approximately to our study region). Since "shared linguistic innovation presupposes a community"; translated from the Slovenian by B.Š.), it follows that heterogeneous dialects presuppose heterogeneous communities or founder populations. Therefore, the linguistic interpretations imply that the southern part of the region under study was originally populated by two founder populations that spoke two heterogeneous Slavic dialects. One, using � glčěti, populated areas along the rivers Mura and Drava, the other one populated areas further west.
## Genetic history
Second, let us turn to genetic history. This scientific field attempts to reconstruct human evolution and the history of human populations using genetic information obtained from either modern or ancient DNA [bib_ref] Genetic History and Identity: The Case of Turkey, Wawruschka [/bib_ref]. DNA has been described as a document containing "messages from the past"and is a proven tool in prehistoric archaeology, e.g., [bib_ref] Genome-wide patterns of selection in 230 ancient Eurasians, Mathieson [/bib_ref] [bib_ref] Genomic insights into the origin of farming in the ancient Near East, Lazaridis [/bib_ref] [bib_ref] Ancient genomes document multiple waves of migration in Southeast Asian prehistory, Lipson [/bib_ref] [bib_ref] The Impact of Ancient Genome Studies in Archaeology, Gokcumen [/bib_ref]. However, there are significant obstacles to the use of modern DNA when it comes to Late Antiquity and the Middle Ages. For example, the historical population-level information that this method reflects is complex and overlapping and should not be understood as representing a direct correspondence between population history and social history [bib_ref] Mapping European Population Movement through Genomic Research, Geary [/bib_ref]. For the study of this time period, ancient DNA or ancient genomic DNA data are more appropriate, e.g., [bib_ref] Ancient genome-wide analyses infer kinship structure in an Early Medieval Alemannic graveyard, O'sullivan [/bib_ref] [bib_ref] Understanding 6th-century barbarian social organization and migration through paleogenomics, Amorim [/bib_ref]. However, ancient DNA data are not available in sufficient quantity to study the expansion of Slavic speakers.
Regardless of the methodological shortcomings, it is agreed that the results of genetic studies on modern DNA are indisputable in terms of providing information on genetic proximity and can contribute to hypotheses about human population history, including Late Antiquity and Early Mediaeval migrations [bib_ref] Mapping European Population Movement through Genomic Research, Geary [/bib_ref] [bib_ref] Understanding 6th-century barbarian social organization and migration through paleogenomics, Amorim [/bib_ref] [bib_ref] Editor's Introduction: The Genetic Challenge to Medieval History and Archaeology, Pohl [/bib_ref] [bib_ref] Why Archaeologists, Historians and Geneticists Should Work Together-and How, Samida [/bib_ref].
The most complete study of modern DNA pertaining to the expansion of Slavic speakers examined all ethnic groups living today who speak Balto-Slavic languages, as well as their neighbors. It concluded that the genetic diversity of today's Slavs was predominantly formed in situ (i.e., the substrate genetic components in the settled areas prevail), with marked differences between West and East Slavs on the one hand and South Slavs on the other. However, there is genetic affinity showing a common ancestry (i.e., a homogeneous genetic substrate inherited from the ancestral population) among the Slavs, which probably demonstrates the historical dispersion of a once uniform population [bib_ref] Genetic Heritage of the Balto-Slavic Speaking Populations: A Synthesis of Autosomal, Mitochondrial..., Kushniarevich [/bib_ref] [bib_ref] Genetics and Slavic Languages, Kushniarevich [/bib_ref]. This was recently confirmed in a review article, which concluded, that the migration of Slavs was accompanied by active assimilation of indigenous European populations [bib_ref] Diversity and Structure of Mitochondrial Gene Pools of Slavs in the Ethnogenetic..., Malyarchuk [/bib_ref].
Looking more closely at the region under study, the variability of the microsatellite loci of the Y chromosome is telling. It shows that the inhabitants of present-day Slovenia are far removed from all other South Slavic populations [bib_ref] Genetic Heritage of the Balto-Slavic Speaking Populations: A Synthesis of Autosomal, Mitochondrial..., Kushniarevich [/bib_ref]. When this was first discovered in an earlier study it was interpreted to "suggests that at least two different migration waves of the Slavs may have reached the Balkans in the early Middle Ages".
Considering only the modern Slovenian population, there is one possible ancestral haplogroup for all Slovenian populations which has the highest frequency along the Mura and Drava rivers. This could indicate that the origin of this ancestral haplogroup was in this area and that it later spread westward.
Thus, population genetic studies show that the southern part of the study region was possibly settled by two separate migrations. The earlier one took place along the Mura and Drava rivers; the later one was to the west of that area.
## Consilience
Interpretations from three scientific fields, using completely different data sets and methods, shows a consilience or convergence of evidence. Archaeological, linguistic, and genetic evidence suggests, with varying degrees of certainty, that there were two separate migrations to the southern part of the region under study: The earlier one along the Mura and Drava rivers, and the later one, which archaeology can locate along the Sava river. Archaeology and genetics suggest that acculturation was the predominant post-migration process. Linguistics and genetics indicate that the migrants were Slavs. In particular, linguistics indicates that the migrants were speakers of Slavic, and genetics confirms that they had a homogeneous genetic substrate inherited from a single ancestral population common only to ethnic groups speaking Slavic today.
Based on consilience, we can define the immigrants who arrived in the Eastern Alps between c. 500 and c. 700 CE with by far the highest reliability to date. They were speakers of Slavic and shared a specific "Slavic" ancestry. Our archaeological analysis places these migrations in space and time with some precision [fig_ref] Fig 5: Time slices from the emerging hot spot analysis for selected areas [/fig_ref].
# Conclusions
The aim of this article was to test the hybrid hypothesis of Slavic migration using archaeological data, with the Alpine Slavs as a case study. The term "migration" in the title was deliberately chosen to be somewhat provocative, as modern historiography and archaeology of the Early Middle Ages tend to downplay the role of physical migrations.
We used selected machine learning methods to analyze an archaeological data set that can be described as Deep Data. Specifically, we used two methods: Time series clustering and a modified emerging hot spot analysis. The former method is directly suitable for archaeology without modification, whereas the latter required two archaeology-specific modifications: The archaeological trend map and the multiscale emerging hot spot analysis.
The results have provided us with an overwhelming quality and quantity of new information. In this article, we have focused on confirming two separate migrations of the Alpine Slavs. Based on the convergence of evidence from archaeology, linguistics, and population genetics, we define the immigrants as Alpine Slavs who were speakers of Slavic and shared specific "Slavic" ancestry. Two founder populations migrated to the Eastern Alps: The first after c. 500 and the second before c. 700 CE.
The identities and ethnicity of the migrants (as defined by modern historiography) are, in our view, beyond the scope of archaeology. The acculturation processes that took place after the migration will be discussed elsewhere. From the available evidence, however, it is clear that the crucial process was cultural spread sensu Heather. We envisage that the number of migrating people was relatively small and more akin to a small group infiltration than a mass migration. The movement itself was a part of it, but the processes that took place afterwards were historically the most important.
Thus, we have achieved the aim of the article, which was to prove the validity of the hybrid hypothesis of Slavic migration with archaeological data. The migration of the Alpine Slavs was a combination of movement of people, cultural diffusion, and language diffusion, all occurring simultaneously. This clearly refutes the hypothesis that only the cultural model or even only the language spread.
While this paper focused on a specific question related to the migration of Slavs, the methods we developed, borrowing and adapting from a variety of disciplines, can be applied to archaeological studies of any period, anywhere that suitable data is available. We hope that these advances will be used beneficially by other scholars and establish a new, practical approach to add to the archaeological arsenal of methodologies.
In the field of machine learning in archaeology and the digital humanities in general, we hope to have shown that, in addition to Big Data, Deep Data also holds great potential.
## Supporting information
[fig] Fig 1: Map of the study area (upper left corner Lat. 48.22015, Lon. 12.35667; lower right corner Lat. 45.29785, Lon. 16.41784). The study area is marked in red and locations of Fig 5A and 5B in black (authors E.L. and B.Š; contains information from OpenStreetMap and OpenStreetMap Foundation, which is made available under the Open Database License; contains information adapted and modified from Copernicus Land Monitoring Service product EU-DEM25, which was produced with funding by the European Union). https://doi.org/10.1371/journal.pone.0274687.g001 [/fig]
[fig] Fig 2: Graphical representations of selected methods: a) space-time cube model (after ESRI); b) comparison of two types of inference: On the left, data from different fields are compiled to draw a unified conclusion (analysis of Pohl's[17] interpretation of our study region as an example), and on the right, consilience.https://doi.org/10.1371/journal.pone.0274687.g002 [/fig]
[fig] Fig 3: Archaeological periodization with time series clustering: LA-Period 1, Late Antiquity; EMA1-Period 2, Early Middle Ages 1; EMA2-Period 3, Early Middle Ages 2. Values on x axis are years CE, values on y axis are unitless and relative. https://doi.org/10.1371/journal.pone.0274687.g003 [/fig]
[fig] Fig 4: Archaeological trend map of the modified categorization of the multiscale emerging hot spot analysis. See S1 Table for the legend (authors E.L. and B.Š; contains information from OpenStreetMap and OpenStreetMap Foundation, which is made available under the Open Database License; contains information adapted and modified from Copernicus Land Monitoring Service product EU-DEM25, which was produced with funding by the European Union). https://doi.org/10.1371/journal.pone.0274687.g004 PLOS ONE [/fig]
[fig] Fig 5: Time slices from the emerging hot spot analysis for selected areas: a) Eastern part of the study area from 450 CE to 650 CE; b) central part of the study area from before 600 CE to after 750 CE (authors E.L. and B.Š; open access raw data sources used: EU-DEM v1.1, https://land.copernicus.eu; OpenStreetMap, https://www.openstreetmap.org). https://doi.org/10.1371/journal.pone.0274687.g005 [/fig]
[fig] S1: Table. Archaeological trend map, detailed description of 16 classes. (DOCX) Appendix. GIS Protocol for multy-scale emerging hot spot analysis. (DOCX) S2 Appendix. Animation of the emerging hot spot analysis time slices from 400 CE to 1100 CE (authors E.L. and B.Š; contains information from OpenStreetMap and OpenStreetMap Foundation, which is made available under the Open Database License; contains information adapted and modified from Copernicus Land Monitoring Service product EU-DEM25, which was produced with funding by the European Union). (GIF) [/fig]
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Iron overload across the spectrum of non‐transfusion‐dependent thalassaemias: role of erythropoiesis, splenectomy and transfusions
Non-transfusion-dependent thalassaemias (NTDT) encompass a spectrum of anaemias rarely requiring blood transfusions. Increased iron absorption, driven by hepcidin suppression secondary to erythron expansion, initially causes intrahepatic iron overload. We examined iron metabolism biomarkers in 166 NTDT patients with b thalassaemia intermedia (n = 95), haemoglobin (Hb) E/b thalassaemia (n = 49) and Hb H syndromes (n = 22). Liver iron concentration (LIC), serum ferritin (SF), transferrin saturation (TfSat) and non-transferrin-bound iron (NTBI) were elevated and correlated across diagnostic subgroups. NTBI correlated with soluble transferrin receptor (sTfR), labile plasma iron (LPI) and nucleated red blood cells (NRBCs), with elevations generally confined to previously transfused patients. Splenectomised patients had higher NTBI, TfSat, NRBCs and SF relative to LIC, than non-splenectomised patients. LPI elevations were confined to patients with saturated transferrin. Erythron expansion biomarkers (sTfR, growth differentiation factor-15, NRBCs) correlated with each other and with iron overload biomarkers, particularly in Hb H patients. Plasma hepcidin was similar across subgroups, increased with >20 prior transfusions, and correlated inversely with TfSat, NTBI, LPI and NRBCs. Hepcidin/SF ratios were low, consistent with hepcidin suppression relative to iron overload. Increased NTBI and, by implication, risk of extra-hepatic iron distribution are more likely in previously transfused, splenectomised and iron-overloaded NTDT patients with TfSat >70%.
## Summary
Non-transfusion-dependent thalassaemias (NTDT) encompass a spectrum of anaemias rarely requiring blood transfusions. Increased iron absorption, driven by hepcidin suppression secondary to erythron expansion, initially causes intrahepatic iron overload. We examined iron metabolism biomarkers in 166 NTDT patients with b thalassaemia intermedia (n = 95), haemoglobin (Hb) E/b thalassaemia (n = 49) and Hb H syndromes (n = 22). Liver iron concentration (LIC), serum ferritin (SF), transferrin saturation (TfSat) and non-transferrin-bound iron (NTBI) were elevated and correlated across diagnostic subgroups. NTBI correlated with soluble transferrin receptor (sTfR), labile plasma iron (LPI) and nucleated red blood cells (NRBCs), with elevations generally confined to previously transfused patients. Splenectomised patients had higher NTBI, TfSat, NRBCs and SF relative to LIC, than non-splenectomised patients. LPI elevations were confined to patients with saturated transferrin. Erythron expansion biomarkers (sTfR, growth differentiation factor-15, NRBCs) correlated with each other and with iron overload biomarkers, particularly in Hb H patients. Plasma hepcidin was similar across subgroups, increased with >20 prior transfusions, and correlated inversely with TfSat, NTBI, LPI and NRBCs. Hepcidin/SF ratios were low, consistent with hepcidin suppression relative to iron overload. Increased NTBI and, by implication, risk of extra-hepatic iron distribution are more likely in previously transfused, splenectomised and iron-overloaded NTDT patients with TfSat >70%.
Keywords: non-transfusion-dependent thalassaemia, iron overload, ineffective erythropoiesis, anaemia.
Non-transfusion-dependent thalassaemias (NTDT) comprise a heterogeneous group of anaemias characterised by impaired production of b-globin and/or a-globin chains, for which ineffective erythropoiesis is the underlying mechanism for anaemia [bib_ref] Hb H disease: clinical course and disease modifiers, Fucharoen [/bib_ref] [bib_ref] Beta-thalassemia, Galanello [/bib_ref] [bib_ref] Identification of erythroferrone as an erythroid regulator of iron metabolism, Harteveld [/bib_ref]. Three main diagnostic subgroups have been identified: b thalassaemia intermedia (b TI), haemoglobin (Hb) E/b thalassaemia and Hb H syndromes [bib_ref] The definition and epidemiology of non-transfusion-dependent thalassemia, Weatherall [/bib_ref]. Management strategies are highly diverse, and may include close observation, sporadic transfusion, splenectomy and Hb F-inducing agents [bib_ref] beta-thalassemia intermedia: a clinical perspective, Musallam [/bib_ref].
Patients with NTDT, as well as other iron-loading anaemias, become iron overloaded mainly because of increased iron absorption [bib_ref] Iron absorption and loading in b-thalassaemia intermedia, Pippard [/bib_ref] [bib_ref] Iron metabolism in thalassemia, Pootrakul [/bib_ref] , which averages 0Á011 mg/kg/day in the absence of transfusion therapy ; sporadic transfusions also directly contribute to further iron accumulation. However, the parameters accounting for variability in iron absorption across the range of NTDT diagnostic subgroups are not well described. In principle, iron accumulation could be increased in line with the degree of anaemia and/or by erythroid expansion, both driving down synthesis of the iron regulatory protein hepcidin and hence increasing dietary iron absorption. Bone-marrow-derived factors associated with ineffective erythropoiesis have been implicated in inhibiting research paper hepcidin synthesis, including growth differentiation factor 15 (GDF-15) in humans [bib_ref] High levels of GDF15 in thalassemia suppress expression of the iron regulatory..., Tanno [/bib_ref] [bib_ref] Iron loading and overloading due to ineffective erythropoiesis, Tanno [/bib_ref] and, more recently, erythroferrone in mice [bib_ref] Identification of erythroferrone as an erythroid regulator of iron metabolism, Harteveld [/bib_ref]. In addition, GDF-11 has recently been identified as a key factor associated with ineffective erythropoiesis in a mouse model of thalassaemia . The same mechanism will also increase the release of recycled iron from the reticuloendothelial system, thus depleting macrophage iron. This typically explains the preferential periportal and hepatocyte iron loading with increased liver iron concentration (LIC) [bib_ref] Liver iron concentrations and urinary hepcidin in bthalassemia, Origa [/bib_ref] [bib_ref] Iron overload in thalassaemia intermedia: reassessment of iron chelation strategies, Taher [/bib_ref] [bib_ref] Optimal management of b-thalassaemia intermedia, Taher [/bib_ref] [bib_ref] Iron loading and overloading due to ineffective erythropoiesis, Tanno [/bib_ref]. Chronic anaemia and hypoxia also induce expression of hypoxia-inducible transcription factors [bib_ref] Iron loading and overloading due to ineffective erythropoiesis, Tanno [/bib_ref] and stimulate erythropoietin (EPO) synthesis, thus inducing erythropoiesis and erythron expansion, which further augments iron absorption.
The THALASSA (Assessment of Exjade in Non-Transfusion-Dependent Thalassaemia) trial was a prospective, randomised, double-blind, placebo-controlled, Phase II clinical trial of deferasirox, which enrolled 166 patients with the three primary diagnostic groups of NTDT and considerable iron burden . The current analyses of baseline data from the large THALASSA database (prior to treatment with deferasirox) examined how erythropoietic and iron metabolism markers inter-relate within NTDT diagnostic subgroups. With the associations between ineffective erythropoiesis, chronic anaemia and gastrointestinal iron absorption that lead to iron loading in patients with NTDT, we hypothesised that LIC would be associated with biomarkers of iron turnover, anaemia and erythroid expansion, particularly in untransfused and unchelated patient subgroups. Baseline markers of iron storage (serum ferritin and LIC) and iron turnover (transferrin saturation [TfSat], non-transferrin-bound iron and labile plasma iron [LPI]) were examined, as well as markers of erythroid expansion (soluble transferrin receptor [sTfR], GDF-15 and nucleated red blood cells . Markers of anaemia (Hb and EPO) were compared between the NTDT subgroups and related to the iron metabolism markers. Plasma hepcidin was measured in all subgroups at baseline and related to markers of iron storage, iron turnover, erythroid expansion, anaemia and hypoxia. The effects of previous management strategies (splenectomy and prior transfusion therapy) on these markers were also examined.
# Methods
## Study design
THALASSA was a multinational, prospective, randomised, double-blind, placebo-controlled, Phase II study (ClinicalTrials.gov number NCT00873041) for which the study design and inclusion/exclusion criteria have been described previously . Briefly, male or female patients aged ≥10 years with NTDT, iron overload (LIC ≥5 mg Fe/g dry weight [dw] by R2 magnetic resonance imaging and serum ferritin >300 lg/l who received no transfusions within 6 months or chelation therapy within 1 month before study entry were included. Patients with previous exposure to deferasirox were excluded, although patients may have received deferoxamine (DFO), deferiprone or a combination of the two. Additional exclusion criteria included Hb S variants of thalassaemia syndromes, active hepatitis B or hepatitis C, cirrhosis, alanine aminotransferase levels >5 9 the upper limit of normal (ULN), serum creatinine >ULN or creatinine clearance ≤60 ml/min on two consecutive measurements, or significant proteinuria (urine protein/urine creatinine ratio >1Á0 mg/mg) on two consecutive measurements. Patients (or parents/guardians) provided written, informed consent prior to enrolment. No patients received any transfusions within 6 months or chelation therapy within 1 month prior to enrolment. The study was conducted in accordance with Good Clinical Practice guidelines and the Declaration of Helsinki and was approved by the local ethics committees of the study sites.
## Assessments
The THALASSA trial included an initial screening period of 4 weeks, composed of two visits at least 14 days apart. During screening, LIC was measured at one visit using a validated R2 MRI technique (FerriScan â , Resonance Health, Claremont, Australia) and adopted as the baseline measurement [bib_ref] Noninvasive measurement and imaging of liver iron concentrations using proton magnetic resonance, St Pierre [/bib_ref]. Serum ferritin levels were measured at both screening visits, and baseline levels were calculated from the mean of the two measurements (with a minimum time lag of 14 days between the two assessments). NRBCs and plasma Hb levels were also measured at both screening visits. TfSat, sTfR, NTBI, LPI, EPO, hepcidin and GDF-15 were measured at baseline (pre-dose). TfSat was directly measured using the urea gel method and NTBI was detected using high-performance liquid chromatography at University College London, London, UK, as described elsewhere [bib_ref] Combined chelation therapy with deferasirox and deferoxamine in thalassemia, Lal [/bib_ref] [bib_ref] Mechanisms of plasma non-transferrin bound iron generation: insights from comparing transfused diamond..., Porter [/bib_ref]. The LPI assay (FeROS TM LPI, Aferrix Ltd., Tel Aviv, Israel) was performed using methods described previously [bib_ref] Labile plasma iron in iron overload: redox activity and susceptibility to chelation, Esposito [/bib_ref]. Plasma hepcidin was determined at King's College London, London, UK. All remaining blood chemistry samples were analysed at a central laboratory.
# Statistical methods
Demographic and other baseline data (including disease characteristics) are summarised descriptively. All analyses were performed for all patients and by diagnostic subgroup: b TI, Hb E/b thalassaemia and Hb H syndromes. Correlations were assessed by Pearson's correlation coefficients and scatter plots, with simple linear regression models; GDF-15 and EPO were analysed as log values for these analyses. Nominal P-values are provided and were considered significant when <0Á05.
# Results
## Baseline demographics and patient characteristics
In total, 166 patients were enrolled. Splenectomy was less frequent in patients with Hb E/b thalassaemia than in patients with b TI. Previous chelation therapy was most frequent in patients with b TI (40%) and least frequent in patients with Hb H syndromes (5%). The proportion of patients who received sporadic or more frequent transfusions and the median number of transfusions were similar across diagnostic subgroups.
## Anaemia and erythropoietic biomarkers
Baseline levels. All patient subgroups were anaemic with elevated EPO levels [fig_ref] Table II: Baseline anaemia, erythroid expansion, iron storage and iron turnover biomarkers in all... [/fig_ref]. However, Hb levels were notably higher and EPO levels lower in patients with Hb H syndromes compared with other diagnostic subgroups, while Hb levels were lowest in patients with Hb E/b thalassaemia. Markers associated with ineffective erythropoiesis (sTfR, GDF-15 and NRBC) were also lowest in patients with Hb H syndromes [fig_ref] Table II: Baseline anaemia, erythroid expansion, iron storage and iron turnover biomarkers in all... [/fig_ref].
Relationships between anaemia, erythropoiesis and iron metabolism. There was a negative correlation between Hb and EPO in the overall population as expected (r = À0Á35; P < 0Á0001; [fig_ref] Table II: Baseline anaemia, erythroid expansion, iron storage and iron turnover biomarkers in all... [/fig_ref]. The EPO-to-Hb relationship was stronger in patients with Hb H syndromes (r = À0Á76; P < 0Á0001) than in patients with Hb E/b thalassaemia (r = À0Á42; P = 0Á0028) and b TI (r = À0Á32; P = 0Á0022). Across all patients, sTfR was negatively related to Hb levels (r = À0Á46; P < 0Á0001; [fig_ref] Fig 1: Correlations between sTfR and [/fig_ref] and positively correlated with EPO (r = 0Á47; P < 0Á0001; [fig_ref] Fig 1: Correlations between sTfR and [/fig_ref]. This was also evident by diagnostic subgroup (Tables SIII, SIV and SV). A negative relationship was present between Hb and NRBCs (r = À0Á46, P = 0Á0016) and between Hb and sTfR (r = À0Á42, P = 0Á0024) in patients with Hb E/b thalassaemia, consistent with more severe anaemias driving increased erythropoiesis; no relationship between Hb and GDF-15 was apparent.
## Iron overload and iron metabolism biomarkers
Baseline levels. Across all diagnostic subgroups, markers of iron storage (LIC and serum ferritin) were raised but similar. Iron turnover biomarkers (TfSat and NTBI) were elevated above normal ranges with lower TfSat and NTBI in patients with Hb H syndromes [fig_ref] Table II: Baseline anaemia, erythroid expansion, iron storage and iron turnover biomarkers in all... [/fig_ref]. Overall, median TfSat was 88% (range 24Á0% to 100Á0%); 34/158 (21Á5%) patients with TfSat measured at baseline had 100% TfSat, which contrasts with transfusion-dependent (TDT) patients, in whom TfSat is typically 100%. LPI was generally not detectable across all subgroups, but reached levels above normal (>0Á4 units) in 22 patients overall (b TI, n = 16; Hb E/b thalassaemia,. Baseline demographics and patient characteristics by underlying disease.
## Characteristic
## All patients
[formula] (n = 166) b TI (n = 95) Hb E/b thalassaemia (n = 49) Hb H syndromes (n = 22) Age at screening, years Mean AE SD 32Á1 AE 12Á0 3 3 Á0 AE 11Á7 2 7 Á7 AE 11Á1 3 7 Á7 AE 12Á4 Median (range) 31Á5 (10-69) 33Á0 (11-69) 27Á0 (10-52) 35Á5 (18-60) Splenectomy, n (%) 88 (53Á0) 67 (70Á5) 18 (36Á7) 3 (13Á6) Transfused previously (sporadic), n (%) 145 (87Á3) 80 (84Á2) 47 (95Á9) 18 (81Á8) Median (range) number of previous transfusions* 8 (1-75) 10 (1-75) 6 (1-47) 6 (1-43) Patients with 1-<10, n (%) 76 (45Á8) 38 (40Á0) 29 (59Á2) 9 (40Á9) Patients with ≥10, † n (%) 65 (39Á2) 39 (41Á1) 18 (36Á7) 8 (36Á4) Previous chelation therapy, ‡ n (%) 44 (26Á5) 37 (38Á9) 6 (12Á2) 1 (4Á5) [/formula]
b TI, b thalassaemia intermedia; Hb, haemoglobin; SD, standard deviation. The proportion of patients who received sporadic or more frequent transfusions was similar across diagnostic subgroups. *Patients did not receive any transfusion in the 6 months prior to study entry and data were missing in four patients. †Data were missing in four patients. ‡Patients did not receive any chelation therapy within 1 month prior to study entry and patients with previous exposure to deferasirox were excluded. n = 5; Hb H syndromes, n = 1). Hepcidin levels were generally within the normal range, although slightly lower in b TI patients than in the other two patient subgroups [fig_ref] Table II: Baseline anaemia, erythroid expansion, iron storage and iron turnover biomarkers in all... [/fig_ref]. The hepcidin/serum ferritin ratio was similar for all diagnostic subgroups [fig_ref] Table II: Baseline anaemia, erythroid expansion, iron storage and iron turnover biomarkers in all... [/fig_ref] and notably less than that described elsewhere in TDT and sickle-cell disease (SCD) patients, using similar methodology for plasma hepcidin measurement [bib_ref] Mechanisms of plasma non-transferrin bound iron generation: insights from comparing transfused diamond..., Porter [/bib_ref]. This is consistent with hepcidin suppression relative to iron overload in NTDT patients across all subgroups. Hepcidin levels were highest in patients with a history of >20 blood transfusions, despite serum ferritin and LIC being no higher in this group.
Relationships between iron storage, iron turnover and hepcidin. Strong correlations were seen between LIC and serum ferritin overall (r = 0Á64; P < 0Á0001) and in all diagnostic subgroups [fig_ref] Table II: Baseline anaemia, erythroid expansion, iron storage and iron turnover biomarkers in all... [/fig_ref] , with similar slopes (data not shown). TfSat correlated with NTBI (r = 0Á84; P < 0Á0001), LIC (r = 0Á37; P < 0Á0001), serum ferritin (r = 0Á35; P < 0Á0001) and LPI (r = 0Á30; P = 0Á0002; [fig_ref] Fig 2: Correlations between TfSat and markers of iron storage [/fig_ref]. The relationship between TfSat and NTBI was continuous [fig_ref] Fig 2: Correlations between TfSat and markers of iron storage [/fig_ref] , whereas the relationship between TfSat and LPI appeared binary or possibly hyperbolic, with LPI absent (except in four cases) when TfSat was <98% [fig_ref] Fig 2: Correlations between TfSat and markers of iron storage [/fig_ref]. This may explain the apparent absence of LPI across diagnostic subgroups, as TfSat was typically <100% [fig_ref] Table II: Baseline anaemia, erythroid expansion, iron storage and iron turnover biomarkers in all... [/fig_ref]. NTBI also correlated with LIC (r = 0Á35; P < 0Á0001; [fig_ref] Fig 3: Correlations between NTBI and markers of iron storage [/fig_ref] and serum ferritin (r = 0Á39; P < 0Á0001; [fig_ref] Fig 3: Correlations between NTBI and markers of iron storage [/fig_ref]. The relationships between iron storage and iron turnover parameters were generally stronger in patients with Hb E/b thalassaemia and Hb H syndromes than in b TI patients [fig_ref] Table II: Baseline anaemia, erythroid expansion, iron storage and iron turnover biomarkers in all... [/fig_ref] and SV). Moderate-to-strong correlations between hepcidin and both TfSat and NTBI were identified (r = À0Á33; P < 0Á0001 and r = À0Á24; P = 0Á0029, respectively; [fig_ref] Fig 4: Correlations between hepcidin and markers of iron turnover [/fig_ref]. There was no correlation between hepcidin and markers of iron storage [fig_ref] Table II: Baseline anaemia, erythroid expansion, iron storage and iron turnover biomarkers in all... [/fig_ref]. This was consistent across underlying diseases [fig_ref] Table II: Baseline anaemia, erythroid expansion, iron storage and iron turnover biomarkers in all... [/fig_ref].
Relationships between erythroid expansion, iron metabolism and hepcidin. Evaluable biomarkers of erythroid expansion generally correlated with each other. Overall, there were strong, positive associations between sTfR and GDF-15 [fig_ref] Fig 1: Correlations between sTfR and [/fig_ref] and between sTfR and NRBCs [fig_ref] Fig 1: Correlations between sTfR and [/fig_ref]. Moderate, yet significant, correlations between markers of erythron expansion (sTfR, NRBC and GDF-15) and iron turnover were also identified [fig_ref] Table II: Baseline anaemia, erythroid expansion, iron storage and iron turnover biomarkers in all... [/fig_ref] : NTBI and sTfR (r = 0Á20; P = 0Á013); NTBI and NRBC (r = 0Á23; P = 0Á0041); TfSat and GDF-15 (r = 0Á17; P = 0Á0402); and TfSat and NRBC (r = 0Á26; P = 0Á0014). These correlations were generally stronger in patients with Hb E/b thalassaemia or Hb H syndromes. Markers of erythron expansion (sTfR, NRBC and GDF-15) were not correlated with those of iron storage in patients as a whole, although relationships were identified in patients with Hb H syndromes: between LIC and sTfR (r = 0Á44; P = 0Á04) and between LIC and GDF-15 (r = 0Á47; P = 0Á027). This may be because most of these patients had not received prior chelation therapy, unlike the other diagnostic subgroups. Hepcidin generally did not correlate with markers of erythroid expansion (GDF-15 or sTfR; [fig_ref] Table II: Baseline anaemia, erythroid expansion, iron storage and iron turnover biomarkers in all... [/fig_ref] except in patients with Hb E/b thalassaemia, where a significant inverse correlation between hepcidin and GDF-15 was seen (r = À0Á3; P = 0Á0084). A significant inverse relationship between hepcidin and NRBCs was also identified overall (r = À0Á21; P = 0Á0081), as well as in patients with Hb E/b thalassaemia [fig_ref] Table II: Baseline anaemia, erythroid expansion, iron storage and iron turnover biomarkers in all... [/fig_ref].
[formula] Hb, g/l 81Á0 (47Á0-140Á0) 84Á0 (47Á0-140Á0) 72Á0 (50Á0-93Á0) 91Á0 (55Á0-106Á0) 113-175 EPO, u/l 101Á0 (18Á3-3405Á0) 108Á0 (18Á3-3405Á0) 115Á0 (19Á1-649Á0) 57Á0 (26Á1-403Á0) 3Á7-31Á5 NRBC,/100 WBC 45Á3 (0Á0-827Á5) 128Á5 (0Á0-827Á5) 18Á0 (0Á0-631Á0) 1Á00 (0Á0-17Á0) 0 sTfR, mg/l 28Á7 (8Á3-64Á3) 27Á9 (8Á3-56Á5) 30Á5 (17Á4-51Á3) 24Á9 (11Á6-64Á3) 1Á9-5Á0 GDF-15,/g dw 12Á1 (2Á6-49Á1) 11Á7 (2Á6-49Á1) 14Á7 (5Á0-42Á1) 11Á3 (6Á2-34Á6) <1Á8 1 TfSat, % 88Á0 (24Á0-100Á0) 88Á5 (24Á0-100Á0) 88Á0 (32Á0-97Á0) 77Á0 (42Á1-98Á3) 20-55 NTBI, lmol/l* 2Á2 (-3Á2-8Á5) 1Á8 (-3Á2-8Á5) 2Á6 (-1Á7-5Á5) 1Á8 (-2Á7-7Á5) 0Á3-1Á5 LPI, LPI units 0Á0 (0Á0-2Á9) 0Á0 (0Á0-2Á9) 0Á0 (0Á0-1Á7) 0Á0 (0Á0-2Á4) 0-0Á4 Hepcidin, nmol/l 4Á4 (0Á1-51Á0) 3Á8 (0Á1-51Á0) 5Á6 (0Á8-38Á7) 5Á0 (1Á2-15Á7) 2Á0-7Á5 Hepcidin/serum ferritin ratioÁ1000 5Á0 (0Á0-50Á0) 4Á0 (0Á0-40Á0) 6Á0 (0Á0-50Á0) 5Á0 (0Á0-50Á0) 23Á2 2 b TI, b [/formula]
## Effects of previous management: transfusion and splenectomy
Prior blood transfusion. The majority of patients received sporadic transfusions prior to enrolment, with a median of eight lifetime transfusion episodes, although the number was highly variable (range 1-75; indicative of a greater degree of ineffective erythropoiesis, as well as increased iron storage and iron turnover biomarkers [fig_ref] Table II: Baseline anaemia, erythroid expansion, iron storage and iron turnover biomarkers in all... [/fig_ref]. LIC and serum ferritin levels were not related to the number of previous transfusions, presumably because of previous chelation therapy. TfSat and sTfR levels progressively increased in patients who received more transfusions. Hepcidin levels were also notably increased in patients who had received >20 previous transfusions. NTBI was generally absent in patients in the absence of prior transfusions (the majority were b TI patients; and serum ferritin differed in splenectomised patients (r = 0Á764; P < 0Á0001 [n = 88]) versus non-splenectomised patients (r = 0Á552; P < 0Á0001 [n = 77]), as the serum ferritin increment was greater in relation to LIC in splenectomised patients [fig_ref] Fig 5: Correlation between serum ferritin and LIC in splenectomised and non-splenectomised patients [/fig_ref]. No differences were noted between LIC and other markers of iron metabolism, TfSat and NTBI (data not shown). However, both NTBI and TfSat were notably higher in splenectomised versus non-splenectomised patients [2Á5 lmol/l (À1Á9-8Á5) vs 0Á6 lmol/l (À3Á2-7Á5) and 91Á0% (43Á0-100Á0) vs 78Á0% (24Á0-98Á3), respectively; [fig_ref] Fig 6: Median AE IQR baseline levels of [/fig_ref]. Splenectomised patients also had higher median (range) GDF-15 levels and NRBCs than non-splenectomised patients [fig_ref] Fig 6: Median AE IQR baseline levels of [/fig_ref] : 10 300 ng/l (1075-53 730) versus 7903 ng/l (689-46 511) and 235Á0/100 white blood cells (WBC) (3Á5-827Á5) vs 4Á0/100 WBC (0Á0-56Á0), respectively. Levels of EPO and Hb were similar: 101Á0 u/l (18Á3-3405Á0) vs 101Á0 u/l (19Á1-1957Á0) and 81Á0 g/l (47Á0-118Á0) vs 81Á0 g/l (55Á0-140Á0), respectively.
# Discussion
To our knowledge, this is the first study to compare a range of biomarkers of iron metabolism and erythropoiesis, including plasma hepcidin, across a range of diagnostic NTDT TfSat and sTfR levels were progressively increased in patients who received more transfusions and hepcidin levels were notably increased in patients who received >20 previous transfusions. Note: Data were missing in four patients. *Negative values can be interpreted as NTBI being absent from the sample.
subgroups. Our findings provide insight into the heterogeneity and similarities across NTDT syndromes, as well as the pathophysiological processes associated with the expanded erythroid and disordered iron metabolism. Patients enrolled in the THALASSA study were not currently receiving regular transfusions or chelation within the past month, but were sufficiently iron overloaded, as defined by LIC and serum ferritin levels, to potentially benefit from iron chelation therapy . Even without a history of transfusions, iron overload occurred across the spectrum of NTDT subgroups.
## Anaemia and hypoxia
Increased markers of anaemia, hypoxia, ineffective erythropoiesis and erythroid expansion (sTfR, GDF-15 and NRBC) were seen across all NTDT subgroups. These markers were distinctly lower in patients with Hb H syndromes and higher in those with b TI. Hb E/b thalassaemia patients were, on average, more anaemic than those with b TI, but EPO levels were similar to b TI patients [fig_ref] Table II: Baseline anaemia, erythroid expansion, iron storage and iron turnover biomarkers in all... [/fig_ref]. This is consistent with the right-shifted oxygen dissociation curve and typically lower Hb F levels in patients with Hb E/b thalassaemia than with b TI [bib_ref] Why are hemoglobin F levels increased in HbE/beta thalassemia?, Rees [/bib_ref]. The negative correlation between Hb levels and EPO across all patient groups supports the known EPO response to anaemia and hypoxia [bib_ref] Haemoglobin E b thalassaemia in Sri Lanka, Premawardhena [/bib_ref] [bib_ref] Optimal management of b-thalassaemia intermedia, Taher [/bib_ref]. The weaker relationship between EPO and Hb in patients with b TI may also be due to increased variability in the presence of Hb F [bib_ref] Serum erythropoietin and erythropoiesis in high-and low-fetal hemoglobin beta-thalassemia intermedia patients, Galanello [/bib_ref] , which was not measured in this study. The strong negative correlation of Hb levels with sTfR [fig_ref] Fig 1: Correlations between sTfR and [/fig_ref] supports the concept that sTfR levels reflect erythroid mass, which increases with greater anaemia. Indeed, sTfR levels have been linked with the incidence of extramedullary erythropoiesis in b TI patients [bib_ref] A useful relationship between the presence of extramedullary erythropoeisis and the level..., Ricchi [/bib_ref]. It is notable that patients with the most previous transfusion episodes (>20) had the lowest Hb values and the highest EPO and sTfR levels, indicative of a group of patients with the greatest demand on the erythron when not regularly transfused.
## Tfsat, ntbi and lpi
Across NTDT subgroups, raised TfSat and NTBI were associated with increased blood transfusion history, splenectomy, increased sTfR, increased NRBCs and lower plasma hepcidin. The strong correlation between TfSat and NTBI, observed for all NTDT subgroups, is consistent with the relationship previously identified in haemochromatosis and thalassaemia major [bib_ref] Determination of non-transferrin-bound iron in genetic hemochromatosis using a new HPLC-based method, Loreal [/bib_ref] [bib_ref] Inflammation and oxidant-stress in beta-thalassemia patients treated with iron chelators deferasirox (ICL670)..., Walter [/bib_ref]. In this study, NTBI was predominantly evident in patients when TfSat exceeded approximately 80%. TfSat is a relatively simple and inexpensive marker and we would recommend its use when NTBI is not routinely available. Prior use of transfusions appeared to be a key factor in the generation of NTBI and TfSat. NTBI was absent in patients without previous transfusions despite median LIC values >7 mg Fe/g dw [fig_ref] Table II: Baseline anaemia, erythroid expansion, iron storage and iron turnover biomarkers in all... [/fig_ref]. This observation suggests that increased macrophage iron may be a key factor in the generation of NTBI because patients overloaded from gastrointestinal iron absorption alone have predominantly periportal hepatocellular iron, whereas transfusion therapy initially loads iron to the macrophage compartment [bib_ref] Liver iron concentrations and urinary hepcidin in bthalassemia, Origa [/bib_ref]. Rapid egress of iron is the key route to transferrin binding and, potentially, NTBI generation [bib_ref] Mechanisms of plasma non-transferrin bound iron generation: insights from comparing transfused diamond..., Porter [/bib_ref]. By contrast, LPI levels showed no obvious relationship to blood transfusion. LPI was not detected in the majority of patients (i.e. levels below the ULN of 0Á4 units), whereas other markers of iron storage and iron turnover were elevated. Raised LPI levels were mainly confined to those patients with 100% TfSat [n = 34 (21Á5%); [fig_ref] Fig 2: Correlations between TfSat and markers of iron storage [/fig_ref] , which explains why, on average, LPI was generally not raised. These findings suggest that LPI is unlikely to be a useful marker of small changes in iron overload in NTDT patients. The binary or hyperbolic relationship of LPI to TfSat [fig_ref] Fig 2: Correlations between TfSat and markers of iron storage [/fig_ref] also contrasts with the continuum seen between NTBI and TfSat [fig_ref] Fig 2: Correlations between TfSat and markers of iron storage [/fig_ref] , suggesting that NTBI and LPI identify different iron species in NTDT. As NTDT patients are typically at lower risk of extra-hepatic iron damage than TDT patients, where LPI is usually raised, this may be consistent with the notion that LPI is a fraction of NTBI that is more likely to distribute extra-hepatically [bib_ref] LPI-labile plasma iron in iron overload, Cabantchik [/bib_ref].
## Relevance of splenectomy
Higher NTBI values in splenectomised than in non-splenectomised patients have been previously reported for patients with b TI [bib_ref] Levels of non-transferrin-bound iron as an index of iron overload in patients..., Taher [/bib_ref]. This could represent a more severe subset of patients who required splenectomy. Further studies of NTBI (and LPI) in patients pre-and post-splenectomy would address this issue. Another explanation, however, is that splenectomy diverts erythrophagocytosis away from the spleen, where any NTBI generated would have been cleared efficiently as a first pass by hepatocytes [bib_ref] Efficient clearance of non-transferrinbound iron by rat liver. Implications for hepatic iron..., Brissot [/bib_ref]. When erythrophagocytosis is diverted to the marrow, NTBI clearance by the liver will occur only after systemic circulation and, hence, with higher NTBI levels and greater extra-hepatic iron uptake.
## Erythrocytic expansion and iron overload
The relationship between markers of iron overload and those of erythron expansion is of mechanistic interest as bone-marrow-derived factors, such as erythroferrone [bib_ref] Identification of erythroferrone as an erythroid regulator of iron metabolism, Harteveld [/bib_ref] , are thought to increase iron absorption through suppression of hepcidin. Unfortunately, many patients with b TI and Hb E/b thalassaemia had received previous transfusion and chelation therapy, thus obscuring such relationships. Only in patients with Hb H syndromes, where transfusion and chelation therapy are rarely used, could relationships between erythron expansion and iron overload be reliably explored. Patients with Hb H syndromes received less chelation (only one patient) and had lower splenectomy rates than other NTDT patient subgroups, even though serum ferritin levels and LIC were increased to a similar degree. Therefore, the correlations between markers of iron overload and erythroid expansion in patients with Hb H syndromes are of particular interest and support a relationship between ineffective erythropoiesis and increased iron absorption across NTDT subgroups. These findings should also be considered in the light of newly discovered markers of ineffective erythropoiesis [e.g. GDF-11 and erythroferrone [bib_ref] Identification of erythroferrone as an erythroid regulator of iron metabolism, Harteveld [/bib_ref] ], which were reported after the completion of the THALASSA trial . Analyses of these biomarkers may provide a more complete interpretation of the relationships between erythrocytic expansion and iron overload in patients with NTDT.
## Plasma hepcidin
Hepcidin is a key molecule in the regulation of iron metabolism. Generally, plasma levels are thought to be increased by iron overload and decreased by erythron expansion. Our findings support a general relationship between hepcidin and Hb. Hepcidin levels were not elevated above normal reference ranges across all NTDT subgroups, despite the presence of iron overload. This is consistent with inhibitory effects from expanded bone marrow [bib_ref] High levels of GDF15 in thalassemia suppress expression of the iron regulatory..., Tanno [/bib_ref] [bib_ref] Identification of erythroferrone as an erythroid regulator of iron metabolism, Harteveld [/bib_ref] outweighing the stimulatory effects of iron overload. It is of interest that hepcidin levels were least suppressed in patients receiving >20 previous transfusion episodes [fig_ref] Table II: Baseline anaemia, erythroid expansion, iron storage and iron turnover biomarkers in all... [/fig_ref]. Another way of examining these competing effects is with the hepcidin/serum ferritin ratio [bib_ref] Transfusion suppresses erythropoiesis and increases hepcidin in adult patients with beta-thalassemia major:..., Pasricha [/bib_ref]. Recently, this ratio (hepcidin *1000/serum ferritin) was found to be higher in controls (ratio = 23Á2) than in patients with thalassaemia major or SCD [bib_ref] Mechanisms of plasma non-transferrin bound iron generation: insights from comparing transfused diamond..., Porter [/bib_ref]. In the NTDT patients from the THALASSA trial, the average ratio was approximately 4Á4 across all NTDT subgroups (4Á0 in b TI; 6Á0 in Hb E/b thalassaemia; 5Á0 in Hb H syndromes). This ratio is consistent with moderate generalised hepcidin suppression when corrected for iron overload, presumably as a consequence of erythroid expansion. In this cross-sectional analysis, we also showed that plasma hepcidin in patients with NTDT was more closely related to markers of iron turnover (TfSat and NTBI) than to iron storage (serum ferritin and LIC). Therefore, single measurements of plasma hepcidin are unlikely to be a useful marker of iron overload in NTDT patients.
## Lic and serum ferritin
The importance of the relationship between the two markers of iron storage, LIC and serum ferritin, lies mainly in the practical management of iron overload in NTDT patients. Increased LIC and serum ferritin levels in NTDT patients are associated with increased morbidities, including vascular, endocrine and bone diseases [bib_ref] Evaluation of the 5 mg/g liver iron concentration threshold and its association..., Musallam [/bib_ref] [bib_ref] Serum ferritin level and morbidity risk in transfusion-independent patients with beta-thalassemia intermedia:..., Musallam [/bib_ref]. Serum ferritin underestimates body iron stores in NTDT patients in comparison with patients with TDT [bib_ref] Serum ferritin underestimates liver iron concentration in transfusion independent thalassemia patients as..., Pakbaz [/bib_ref] [bib_ref] Correlation of liver iron concentration determined by R2 magnetic resonance imaging with..., Taher [/bib_ref] [bib_ref] Heterogeneity of hemoglobin H disease in childhood, Lal [/bib_ref]. However, clinically relevant serum ferritin thresholds have been identified [bib_ref] Serum ferritin level and morbidity risk in transfusion-independent patients with beta-thalassemia intermedia:..., Musallam [/bib_ref] [bib_ref] Defining serum ferritin thresholds to predict clinically relevant liver iron concentrations for..., Taher [/bib_ref] , providing a convenient and inexpensive method of assessing iron overload and tailoring iron chelation therapy when there is restricted access to MRI technology. In the present study, the relationship between LIC and serum ferritin was consistent across all NTDT subgroups. However, the relationship between these two parameters differed between splenectomised and nonsplenectomised patients as serum ferritin levels were increased relative to LIC in splenectomised patients. A mechanistic interpretation requires consideration. The reported lower serum ferritin levels, relative to LIC, in NTDT versus TDT patients is probably secondary to early iron deposition in periportal hepatocytes rather than macrophages [bib_ref] Liver iron concentrations and urinary hepcidin in bthalassemia, Origa [/bib_ref]. As serum ferritin at values <3000 lg/l are mainly derived from macrophages [bib_ref] Binding of human serum ferritin to concanavalin A, Worwood [/bib_ref] , the hepatocellular accumulations are relatively 'invisible' to the assay for serum ferritin. It is likely that splenectomised patients received more transfusions prior to splenectomy than nonsplenectomised patients, thereby leading to greater iron in the macrophage system and, hence, higher serum ferritin levels relative to LIC. Despite these caveats, serum ferritin levels show the closest relationship to LIC of any of the biomarkers examined. Indeed, the other biomarkers, while being of mechanistic interest, do not correlate with LIC closely enough to justify clinical decisions about chelation therapy to be made on the basis of their levels.
# Conclusions
These analyses add insight into the complex interactions between anaemia, ineffective erythropoiesis and iron metabolism, as well as previous clinical management across the clinical spectrum of NTDT patients. Importantly, we have observed increased markers of iron overload across all underlying diagnostic syndromes, confirming significant iron burden in NTDT patients who may, therefore, benefit from optimised iron chelation therapy. A possible relationship of iron overload to the degree of erythroid expansion was seen in patients with Hb H syndromes but was obscured in other subgroups, most likely due to prior blood transfusions and chelation therapy. These Hb H patients were predominantly untransfused and we suggest that in such patients, biomarkers of erythron expansion could, in principle, be predictive of those patients at most risk of developing iron overload. Future studies should aim to validate these relationships in larger patient groups who have not received transfusion or chelation therapy. Our evidence suggests that NTDT patients are at a low risk of NTBI-or LPI-mediated extra-hepatic damage unless blood transfusions exceed 20 episodes and transferrin is 100% saturated. Therefore, the history of transfusional iron intake, as well as markers or iron overload, should be considered when assessing patients for iron chelation therapy. The findings with hepcidin are of mechanistic interest, particularly the inverse relationship between hepcidin, TfSat, NTBI and LPI, suggesting that high hepcidin levels may limit TfSat, NTBI and LPI levels in NTDT patients and, hence, the extrahepatic distribution of iron. However, our findings suggest that hepcidin measurement would not add significantly to the routine management of NTDT patients. The study patients, while representing a wide diagnostic spectrum, may not be fully representative of the full range of NTDT syndromes in the 'real world,' because less iron-overloaded patients were excluded from this study. Other parameters would be of interest for future studies, such as erythroferrone, a newly described erythroid regulatory hormone of hepcidin. However, this study is the most comprehensive examination of factors associated with iron overload across the NTDT spectrum. Author contributions JB Porter, MD Cappellini, A Kattamis, V Viprakasit and AT Taher served as investigators on this trial, enrolling patients. They also served as Study Steering Committee members overseeing the conduct of the trial, from study design to analysis plan and data interpretation. KM Musallam contributed to the analysis, interpretation and reporting of the trial data. Z Zhu served as the trial statistician. All authors contributed to data interpretation, reviewed and provided their comments on this manuscript and approved the final version.
## Conflicts of interest
## Supporting information
Additional Supporting Information may be found in the online version of this article:. Overview of anaemia, erythron expansion and iron metabolism parameters examined in this study. [fig_ref] Table II: Baseline anaemia, erythroid expansion, iron storage and iron turnover biomarkers in all... [/fig_ref]. Summary of all correlations between biomarkers of iron overload, anaemia and erythroid expansion in all NTDT patients (n = 166). [fig_ref] Table II: Baseline anaemia, erythroid expansion, iron storage and iron turnover biomarkers in all... [/fig_ref]. Summary of all correlations between biomarkers of iron overload, anaemia and erythroid expansion in patients with b TI (n = 95). Summary of all correlations between biomarkers of iron overload, anaemia and erythroid expansion in patients with Hb E/b thalassaemia (n = 49). . Summary of all correlations between biomarkers of iron overload, anaemia and erythroid expansion in patients with Hb H syndromes (n = 23)
[fig] Fig 1: Correlations between sTfR and (A) Hb (B) NTBI (C) GDF-15 and (D) NRBCs. Evaluable biomarkers of erythroid expansion generally correlated with each other. The strong negative correlation of Hb levels with sTfR supports the concept that sTfR levels reflect erythroid mass, which increases with greater anaemia. GDF-15, growth differentiation factor-15; Hb, haemoglobin; NRBC, nucleated red blood cells; NTBI, non-transferin-bound iron; sTfR, soluble transferrin receptor. [/fig]
[fig] Fig 2: Correlations between TfSat and markers of iron storage: (A) LIC and (B) serum ferritin; and markers of iron turnover: (C) NTBI and (D) LPI. The relationship between TfSat and NTBI was continuous, whereas the relationship between TfSat and LPI appeared binary. b TI, b thalassaemia intermedia; dw, dry weight; Hb, haemoglobin; LIC, liver iron concentration; LPI, labile plasma iron; NTBI, non-transferrin-bound iron; TfSat, transferrin saturation. [/fig]
[fig] Fig 3: Correlations between NTBI and markers of iron storage: (A) LIC and (B) serum ferritin. Strong correlations were noted between NTBI as a marker of iron turnover and LIC and serum ferritin, markers of iron storage. dw, dry weight; LIC, liver iron concentration; NTBI, nontransferrin-bound iron. Erythropoiesis and Iron Metabolism Markers in NTDT ª 2016 The Authors. British Journal of Haematology published by John Wiley & Sons Ltd. [/fig]
[fig] Fig 4: Correlations between hepcidin and markers of iron turnover: (A) TfSat and (B) NTBI. Hepcidin correlated with markers of iron turnover, but not markers of iron storage. b TI, b thalassaemia intermedia; dw, dry weight; NTBI, non-transferrin-bound iron; TfSat, transferrin saturation.Table III. Iron and haematological parameters in all patients by number of previous transfusions. dry weight; EPO, erythropoietin; GDF-15, growth differentiation factor 15; Hb, haemoglobin; LIC, liver iron concentration; LPI, labile plasma iron; NRBC, nucleated red blood cell; NTBI, non-transferrin-bound iron; sTfR, soluble transferrin receptor; TfSat, transferrin saturation; WBC, white blood cells. [/fig]
[fig] Fig 5: Correlation between serum ferritin and LIC in splenectomised and non-splenectomised patients. The relationship between LIC and serum ferritin differed in splenectomised patients, as the serum ferritin increment was greater in relation to LIC. dw, dry weight; LIC, liver iron concentration. Erythropoiesis and Iron Metabolism Markers in NTDT ª 2016 The Authors. British Journal of Haematology published by John Wiley & Sons Ltd. [/fig]
[fig] Fig 6: Median AE IQR baseline levels of (A) NTBI, (B) TfSat, (C) GDF-15 and (D) NRBCs in splenectomised and non-splenectomised patients. Splenectomised patients generally had higher levels of NTBI, TfSat, GDF-15 and NRBCs. GDF-15, growth differentiation factor 15; IQR, interquartile range; NRBC, nucleated red blood cell; NTBI, non-transferrin-bound iron; TfSat, transferrin saturation. [/fig]
[table] Table II: Baseline anaemia, erythroid expansion, iron storage and iron turnover biomarkers in all patients and by diagnostic subgroup. [/table]
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Natural Polymeric Materials: A Solution to Plastic Pollution from the Agro-Food Sector
Citation: Acquavia, M.A.; Pascale, R.; Martelli, G.; Bondoni, M.; Bianco, G. Natural Polymeric Materials: A Solution to Plastic Pollution from the Agro-Food Sector. Polymers 2021, 13, 158. https://doi.
# Introduction
Over the last 50 years, the global production of synthetic plastics, which are carbonbased polymers such as polypropylene, polyethylene, polyvinyl chloride, polystyrene, nylon, and polycarbonate, has continuously increased and it is expected to double in the next 20 years. Approximately 360 million tons of plastics were produced in 2018 and 17% of it has been produced in Europe. Conventional plastics play a pivotal role in modern society, since they can be readily manufactured to an expanding range of products used in civil and industrial applications thanks to their light weight, flexibility, and durability [bib_ref] Ecologically derived waste management of conventional plastics, Lee [/bib_ref]. However, the huge amount of plastic waste production is one of the most-faced issues over the world both for environmental problems and human health threat [bib_ref] Plastic and Human Health: A Micro Issue?, Wright [/bib_ref].
Traditional plastics are almost completely derived from petrochemicals, meaning that fossil feedstocks are used in their production [bib_ref] Environmental factors influencing landfill gas biofiltration: Lab scale study on methanotrophic bacteria..., Amodeo [/bib_ref] [bib_ref] CO 2 and N 2 O from water resource recovery facilities: Evaluation..., Caniani [/bib_ref]. Around 80% of nonfuel chemicals produced by the petrochemical industry are sold for the manufacturing of plastics, thus contributing to environmental pollution as the extraction of oil and gas, particularly hydraulic fracturing for natural gas, releases an array of toxic substances into the air and water, often in significant volumes [bib_ref] CO 2 and N 2 O from water resource recovery facilities: Evaluation..., Caniani [/bib_ref] [bib_ref] N 2 O and CO 2 Emissions from secondary settlers in WWTPs:..., Caivano [/bib_ref] [bib_ref] Disinfection unit of water resource recovery facilities: Critical issue for N2O Emission, Caivano [/bib_ref] [bib_ref] Validation of an analytical method for simultaneous high-precision measurements of greenhouse gas..., Pascale [/bib_ref]. Furthermore, at the end of the 20th century, plastics were found to be persistent pollutants in many environmental niches, since they are largely non-biodegradable; therefore, once they reach the environment in the form of macro or microplastics, contamination and accumulation in food chains through agricultural land, beginning cellulose recovered from lignocellulosic biomass is converted into xylitol and sorbitol, which are easily hydrolyzed to EG in the presence of several mono-and bimetallic phosphide catalysts [bib_ref] Hydrogenolysis of polyalcohols in the presence of metal phosphide catalysts, Soták [/bib_ref] [bib_ref] One-pot catalytic conversion of cellulose into polyols with Pt/CNTs catalysts, Yang [/bib_ref]. Moreover, bio-ethanol derived from sugar cane or corn stover and glycerol, as a co-product of biodiesel, have been used as a feedstock to produce EG [bib_ref] Synthesis of ethylene glycol and terephthalic acid from biomass for producing PET, Pang [/bib_ref]. The production of terephtalic acid by green chemistry processes based on the use of chemical precursors extracted from corn, sugar beet, or orange peel, i.e., isobutanol, 5-hydroxymethylfurfural, and limonene, respectively, is used to a smaller extent [bib_ref] Terephthalic acid from renewable sources: Early-stage sustainability analysis of a bio-PET precursor, Volanti [/bib_ref]. (1) Drop-in bioplastics (i.e., bio-based or partly biobased but non-biodegradable plastics), [bib_ref] Ecologically derived waste management of conventional plastics, Lee [/bib_ref] bio-based non drop-in bioplastics, and (3) fossil based non drop-in bioplastics. According to their origin, non-drop-ins (i.e., biodegradable plastics) are divided into (i) vegetal or animal derived polysaccharides and proteins, (ii) polymers from microorganism, (iii) polymers from biotechnology, and (iv) blends of biopolymers and commercial polyesters [bib_ref] Study of Bio-plastics As Green & Sustainable Alternative to Plastics, Reddy [/bib_ref].
While drop-ins are well known on the market, non-drop-in bioplastics, i.e., plastics that are biodegradable bio-based or based on fossil sources, are alternative materials usually used in niche fields, for example for food services, agriculture, or biomedical applications; therefore, their trade has been emerging only in recent years [bib_ref] Bioplastics Tipping Point: Drop-in or non-drop-in?, De Almeida Oroski [/bib_ref] [bib_ref] Polyhydroxyalkanoates: A way to sustainable development of bioplastics, Jain [/bib_ref] [bib_ref] Poly(hydroxyalkanoates) for Food Packaging: Application and Attempts towards Implementation, Koller [/bib_ref]. These biodegradable polymers can be classified, according to their origin, into four major categories, namely (i) agro-polymers, (ii) polymers from microorganism, (iii) polymers from biotechnology, and (iv) blend of biopolymer and commercial polyesters [fig_ref] Figure 1: Scheme of bioplastics classification [/fig_ref].
Starch, cellulose, pectin as well as animal and vegetable proteins, such as casein and gluten, are well known for being feedstock of agro-polymers based bioplastics [bib_ref] Direct transformation of edible vegetable waste into bioplastics, Bayer [/bib_ref] [bib_ref] Water Aided Fabrication of Whey and Albumin Plastics, Sharma [/bib_ref] [bib_ref] Bioplastics based on wheat gluten processed by extrusion, Jiménez-Rosado [/bib_ref]. Starch, cellulose, and pectin are polysaccharides that can be extracted from several vegetables and fruits (potato, corn, rice, tapioca, apple) and they are used mainly to produce packaging materials. Often, protein additives are used in order to fabricate materials with novel or improved technological properties. In fact, due to the difference between the elemental composition of proteins (covalent bonds between hundreds of amino acids) and polysaccharides (covalent bonds between monosaccharides with ramifications), their mixtures can evidence a wide variety of two-and three-dimensional structures with different physicochemical and rheological properties [bib_ref] Thermo-mechanical and hydrophilic properties of polysaccharide/gluten-based bioplastics, Zárate-Ramírez [/bib_ref].
In addition, there are a lot of polymers that could be produced by a range of microorganisms, cultured under different nutrient and environmental conditions [bib_ref] Bioplastics from microorganisms, Luengo [/bib_ref]. Polyhydroxyalkanoates (PHAs), for example, are linear thermoplastic polymers, with hydroxyalkanoic acid as a monomer unit, which can be synthesized intracellularly as insoluble (1) Drop-in bioplastics (i.e., bio-based or partly bio-based but nonbiodegradable plastics), [bib_ref] Ecologically derived waste management of conventional plastics, Lee [/bib_ref] bio-based non drop-in bioplastics, and (3) fossil based non drop-in bioplastics. According to their origin, non-drop-ins (i.e., biodegradable plastics) are divided into (i) vegetal or animal derived polysaccharides and proteins, (ii) polymers from microorganism, (iii) polymers from biotechnology, and (iv) blends of biopolymers and commercial polyesters [bib_ref] Study of Bio-plastics As Green & Sustainable Alternative to Plastics, Reddy [/bib_ref].
While drop-ins are well known on the market, non-drop-in bioplastics, i.e., plastics that are biodegradable bio-based or based on fossil sources, are alternative materials usually used in niche fields, for example for food services, agriculture, or biomedical applications; therefore, their trade has been emerging only in recent years [bib_ref] Bioplastics Tipping Point: Drop-in or non-drop-in?, De Almeida Oroski [/bib_ref] [bib_ref] Polyhydroxyalkanoates: A way to sustainable development of bioplastics, Jain [/bib_ref] [bib_ref] Poly(hydroxyalkanoates) for Food Packaging: Application and Attempts towards Implementation, Koller [/bib_ref]. These biodegradable polymers can be classified, according to their origin, into four major categories, namely (i) agro-polymers, (ii) polymers from microorganism, (iii) polymers from biotechnology, and (iv) blend of biopolymer and commercial polyesters [fig_ref] Figure 1: Scheme of bioplastics classification [/fig_ref].
Starch, cellulose, pectin as well as animal and vegetable proteins, such as casein and gluten, are well known for being feedstock of agro-polymers based bioplastics [bib_ref] Direct transformation of edible vegetable waste into bioplastics, Bayer [/bib_ref] [bib_ref] Water Aided Fabrication of Whey and Albumin Plastics, Sharma [/bib_ref] [bib_ref] Bioplastics based on wheat gluten processed by extrusion, Jiménez-Rosado [/bib_ref]. Starch, cellulose, and pectin are polysaccharides that can be extracted from several vegetables and fruits (potato, corn, rice, tapioca, apple) and they are used mainly to produce packaging materials. Often, protein additives are used in order to fabricate materials with novel or improved technological properties. In fact, due to the difference between the elemental composition of proteins (covalent bonds between hundreds of amino acids) and polysaccharides (covalent bonds between monosaccharides with ramifications), their mixtures can evidence a wide variety of two-and three-dimensional structures with different physicochemical and rheological properties [bib_ref] Thermo-mechanical and hydrophilic properties of polysaccharide/gluten-based bioplastics, Zárate-Ramírez [/bib_ref].
In addition, there are a lot of polymers that could be produced by a range of microorganisms, cultured under different nutrient and environmental conditions [bib_ref] Bioplastics from microorganisms, Luengo [/bib_ref]. Polyhydroxyalkanoates (PHAs), for example, are linear thermoplastic polymers, with hydroxyalkanoic acid as a monomer unit, which can be synthesized intracellularly as insoluble cytoplasmic inclusions by heterotrophic bacteria, such as Cupriavidus necator [bib_ref] Impact of different by-products from the biodiesel industry and bacterial strains on..., Ribeiro [/bib_ref] [bib_ref] The influence of crude glycerin and nitrogen concentrations on the production of..., Campos [/bib_ref] , recombinant Escherichia coli [bib_ref] A microbial polyhydroxyalkanoates (PHA) based bio-and materials industry, Chen [/bib_ref] , and also by photoautotrophic microorganisms like microalgae [bib_ref] Microalgae as source of polyhydroxyalkanoates (PHAs) -A review, Costa [/bib_ref]. Their synthesis occurs in the presence of an excess of carbon, when other essential nutrients such as oxygen, phosphorus, or nitrogen are limited; after their extraction from cell cultures, they can be processed in a similar way to that of polypropylene, including extrusion and injection molding, obtaining a material with similar properties as well.
On the other hand, bacterial microorganisms can also be used to produce, in a biotechnological approach, biodegradable polymers through the fermentation of carbohydrates obtained from agricultural by-products such as starchy substances as corn, wheat, or sugar and corn starch. Poly Lactic Acid (PLA)-based bioplastics are obtained from a fermentative process that involves conversion of corn, or other carbohydrate sources into dextrose, followed by fermentation/conversion into lactic acid [bib_ref] Study of Bio-plastics As Green & Sustainable Alternative to Plastics, Reddy [/bib_ref]. Thus, lactic acid is isolated and polymerized to yield a low molecular weight, brittle polymer whose chain length could be increased by using external coupling agents.
The last group of biodegradable materials is represented by blends of biopolymers and polymers obtained by chemical synthesis from fossil resources [bib_ref] Development of bioplastic materials: From rapeseed oil industry by products to added-value..., Delgado [/bib_ref] [bib_ref] Influence of processing parameters on the impact strength of biocomposites: A statistical..., Muthuraj [/bib_ref] [bib_ref] The quest for high glass transition temperature bioplastics, Nguyen [/bib_ref]. Polymers blending is a technique that allows to modify the properties of a material using a conventional technology at low cost. In this way, biodegradable polyesters such as Poly CaproLactone (PCL), which is obtained by the condensation of 6-hydroxycaproic acid or through the ring opening polymerization of ε-caprolactone [bib_ref] Synthesis of polycaprolactone: A review, Labet [/bib_ref] , could be easily used to improve mechanical properties of natural polymers as starch, conferring them a better water resistance due to its hydrophobicity [bib_ref] Properties of thermoplastic blends: Starch-polycaprolactone, Averous [/bib_ref].
Nowadays, although with different percentages, all bioplastics are used in a wide range of sectors: from packaging, catering products, consumer electronics, automotive, agriculture/horticulture, and toys to textile fields. The field of application of a given bioplastic material is clearly dictated by its mechanical properties as well as by its biobased content or its biodegradability. These characteristics are evaluated before bioplastics are promoted on the market.
Moreover, to produce functional materials with biological properties, in the case of bioplastics obtained from fruits and vegetable wastes, a metabolic characterization of the raw material is needed. Among the analytical techniques used to this aim, both chromatography and the mass spectrometry are critical as they allow both targeted and untargeted characterization of the main classes of metabolites occurring in a given matrix [bib_ref] Acylated glucosinolates with diverse acyl groups investigated by high resolution mass spectrometry..., Bianco [/bib_ref] [bib_ref] A three-factor Doehlert matrix design in optimising the determination of octadecyltrimethylammonium bromide..., Cataldi [/bib_ref] [bib_ref] Fatty acid neutral losses observed in tandem mass spectrometry with collision-induced dissociation..., Zianni [/bib_ref] [bib_ref] Dibenzo-p-dioxins and dibenzofurans in human breast milk collected in the area of..., Bianco [/bib_ref] [bib_ref] Identification of unsaturated N-acylhomoserine lactones in bacterial isolates of Rhodobacter sphaeroides by..., Cataldi [/bib_ref] [bib_ref] Mass spectrometry-based phytochemical screening for hypoglycemic activity of Fagioli di Sarconi beans..., Pascale [/bib_ref] [bib_ref] Determination of soyasaponins in Fagioli di Sarconi beans (Phaseolus vulgaris L.) by..., Bianco [/bib_ref] [bib_ref] Investigation of the Effects of Virgin Olive Oil Cleaning Systems on the..., Pascale [/bib_ref] [bib_ref] Profiling of quercetin glycosides and acyl glycosides in sun-dried peperoni di Senise..., Pascale [/bib_ref] [bib_ref] Validation of a liquid chromatography coupled with tandem mass spectrometry method for..., Pascale [/bib_ref] [bib_ref] Coceth sulfate characterization by electrospray ionization tandem mass spectrometry, Onzo [/bib_ref].
## Bioplastics bio-based content and biodegradability
Both bio-based and either biodegradable plastic materials have become the world's most widely choice among bioplastic materials as their production has a low environmental cost compared to traditional plastic ones [bib_ref] ScienceDirect Sustainability of bioplastics: Opportunities and challenges, Thakur [/bib_ref]. To promote the diffusion of either bio-based or biodegradable plastic materials, the Public Procurement Working Group of the European Commission's Expert Group for Bio-based Products published 15 recommendations in order to enable procurement policies to embrace eco-friendly materials. Due to emerging trade of this type of materials, it is necessary to establish a labelling harmonization, as well as the existence of standards and test methods to define and measure properties and characteristics like bio-based content, biodegradability, and other attributes unique to ready-to-market products.
The bio-based content of a material is the amount of the biomass-derived carbon, as compared to its total organic carbon content (TOC). The carbon content of biobased materials is determined independently and unequivocally as reported in international standard methods of the American Society for Testing and Materials (ASTM) and of the International Organization for Standardization (ISO). In detail, ASTM D6866-20 and ISO 16620-2 methods report radiocarbon analysis as the technique to determine the bio-based content of solid, liquid, and gaseous samples. The employment of the radiocarbon dating method is based on the significative difference in 14 C isotopic signature between the fossil derived ( 14 C-free) and the biomass derived ( 14 C-including) materials. In detail, the presence of 14 C in the bio-based materials is due to the fact that 14 C containing carbon dioxide formed in the atmosphere, participates in the photosynthetic processes from which Polymers 2021, 13, 158 6 of 39 the biomass derives. Thus, the 14 C content of biomass derived materials is the result, in a first approximation, of 14 C atmospheric levels [bib_ref] Determination of the Biobased Content in Plastics by Radiocarbon, Quarta [/bib_ref]. [bib_ref] Conservation and Recycling Life cycle assessments of biodegradable, commercial biopolymers-A critical review, Yates [/bib_ref] C measurements could be done by using Accelerator Mass Spectrometry (AMS) along with Isotope Ratio Mass Spectrometry (IRMS) or by using Liquid Scintillation Counting (LSC) techniques (ASTM International, 2020). In order to define a bioplastic as bio-based, a biomass-derived carbon content not less than 25% is required.
Another key bioplastic property to be measured is the biodegradability [bib_ref] Biodegradation of bioplastics in natural environments, Emadian [/bib_ref] , which refers to the ability of a material to decompose after interactions with biological elements. The biodegradation of polymers involves three steps: bio-deterioration, bio-fragmentation, and assimilation [fig_ref] Figure 2: The main three steps through which the biodegradation of polymers occurs [/fig_ref] [bib_ref] Polymer biodegradation: Mechanisms and estimation techniques, Lucas [/bib_ref]. Bio-deterioration is the modification of mechanical, chemical, and physical properties of the polymer due to the growth of microorganisms on or inside the surface of the polymers. In the bio-fragmentation step, microorganisms fragment polymers in oligomers and monomers, which, in the next assimilation step, are available as their carbon, energy, and nutrient sources finally with CO 2 , water, and biomass as by-products [bib_ref] Biodegradation of bioplastics in natural environments, Emadian [/bib_ref]. It should be pointed out that only specific microorganisms could degrade a given type of bioplastic. It has been reported that PCL can be degraded by bacteria isolates that exist in deep sea sediments, but these isolates are incapable of degrading other types of bioplastics, such as PLA, PHB, and PBS; however, there exist composting bacteria capable of degrading the latter [bib_ref] Biodegradation of bioplastics in natural environments, Emadian [/bib_ref].
derived ( C -free) and the biomass derived ( C-including) materials. In detail, the presence of 14 C in the bio-based materials is due to the fact that 14 C containing carbon dioxide formed in the atmosphere, participates in the photosynthetic processes from which the biomass derives. Thus, the 14 C content of biomass derived materials is the result, in a first approximation, of 14 C atmospheric levels [bib_ref] Determination of the Biobased Content in Plastics by Radiocarbon, Quarta [/bib_ref]. 14 C measurements could be done by using Accelerator Mass Spectrometry (AMS) along with Isotope Ratio Mass Spectrometry (IRMS) or by using Liquid Scintillation Counting (LSC) techniques (ASTM International, 2020). In order to define a bioplastic as bio-based, a biomass-derived carbon content not less than 25% is required.
Another key bioplastic property to be measured is the biodegradability [bib_ref] Biodegradation of bioplastics in natural environments, Emadian [/bib_ref] , which refers to the ability of a material to decompose after interactions with biological elements. The biodegradation of polymers involves three steps: bio-deterioration, bio-fragmentation, and assimilation [fig_ref] Figure 2: The main three steps through which the biodegradation of polymers occurs [/fig_ref] [bib_ref] Polymer biodegradation: Mechanisms and estimation techniques, Lucas [/bib_ref]. Bio-deterioration is the modification of mechanical, chemical, and physical properties of the polymer due to the growth of microorganisms on or inside the surface of the polymers. In the bio-fragmentation step, microorganisms fragment polymers in oligomers and monomers, which, in the next assimilation step, are available as their carbon, energy, and nutrient sources finally with CO2, water, and biomass as by-products [bib_ref] Biodegradation of bioplastics in natural environments, Emadian [/bib_ref]. It should be pointed out that only specific microorganisms could degrade a given type of bioplastic. It has been reported that PCL can be degraded by bacteria isolates that exist in deep sea sediments, but these isolates are incapable of degrading other types of bioplastics, such as PLA, PHB, and PBS; however, there exist composting bacteria capable of degrading the latter [bib_ref] Biodegradation of bioplastics in natural environments, Emadian [/bib_ref]. The biodegradation of bio-plastic materials is highly dependent on their chemical structures. Generally, polymers with a shorter chain, more amorphous parts, and less complex formula are more susceptible to biodegradation by microorganisms [bib_ref] Methodologies to assess biodegradation of bioplastics during aerobic composting and anaerobic digestion:..., Ruggero [/bib_ref]. The presence of additives could influence the biodegradability of a matrix. As an example, polypyrrole, the archetype of polymers integrated in biosensing devices for biomedical applications, can acquire enhanced biodegradability if grafted onto cellulose chains, thus forming biocomposite [bib_ref] Assay of serum cholinesterase activity by an amperometric biosensor based on a..., Ciriello [/bib_ref] [bib_ref] A novel approach for the selective analysis of L-lysine in untreated human..., Ciriello [/bib_ref]. Moreover, the pH, temperature, and the oxygen content of the environment in which the polymers are placed or disposed of, could be key factors for The biodegradation of bio-plastic materials is highly dependent on their chemical structures. Generally, polymers with a shorter chain, more amorphous parts, and less complex formula are more susceptible to biodegradation by microorganisms [bib_ref] Methodologies to assess biodegradation of bioplastics during aerobic composting and anaerobic digestion:..., Ruggero [/bib_ref]. The presence of additives could influence the biodegradability of a matrix. As an example, polypyrrole, the archetype of polymers integrated in biosensing devices for biomedical applications, can acquire enhanced biodegradability if grafted onto cellulose chains, thus forming biocomposite [bib_ref] Assay of serum cholinesterase activity by an amperometric biosensor based on a..., Ciriello [/bib_ref] [bib_ref] A novel approach for the selective analysis of L-lysine in untreated human..., Ciriello [/bib_ref]. Moreover, the pH, temperature, and the oxygen content of the environment in which the polymers are placed or disposed of, could be key factors for their biodegradation [bib_ref] Aerobic and anaerobic biodegradability of polymer films and physico-chemical characterization, Massardier-Nageotte [/bib_ref] [bib_ref] Compostability of bioplastic packaging materials: An overview, Kale [/bib_ref]. For example, oxidative-degradable polymers accelerate their decomposition under the effect of oxidation through heat and/or UV light. UV radiation can disrupt polymer chains, since the radiation can be absorbed by oxygencontaining components to initiate a primary degradation; these polymers are known as photodegradable polymers. During photodegradation, both molar mass and crystal structure are affected. The plastics that have the capacity to biodegrade by hydrolytic mechanisms such as biopolymers made of cellulose, starch, and polyesters such as PHA are known ad hydro-biodegradable bioplastics.
To date, a wide variety of methods for measuring the biodegradability of polymeric biomaterials have been currently developed and most of them are in agreement with ASTM, ISO, and European Standards (EN) standard methods in terms of environmental conditions, timings, and scales of the tests. Overall, all methods are focused on an indirect measure of degradation process, such as oxygen consumption or biogas generation (CO 2 ) by measuring differences of pressure in the test flasks and carbon dioxide production. A biodegradation level higher than 90% in comparison with cellulose (positive standard) in 180 days, under conditions of controlled composting measured through respirometric methods has been established by the European Norm EN 13,432 as the level for a material/product to be defined as biodegradable and compostable. In addition, a disintegration level higher than 90% in three months and the respective ecotoxicity and chemical safety criteria should be kept. Then, only when the products meet the EN 13,432 standard criteria can the wording "biodegradable" be reported on the packaging label.
The biodegradation of bioplastics has beenextensively investigated in soil and compost environments, where they mainly showed high degradability [bib_ref] Biodegradation of bioplastics in natural environments, Emadian [/bib_ref]. Anyway, the conditions of the experiments conducted to study the bioplastics biodegradability are highly variable, and to make a clear comparison among them is difficult. The experiments carried out in compost or in anaerobic digestion environments show a biodegradability over 50% in 65 and 68%, respectively. For those carried out in aquatic environments, this share is 44%, and for experiments carried out in soil, it is 33% of the cases.
It should be pointed out that, in addition to increasing bio-based content and biodegradability, bioplastics intrinsic properties often need to be improved to meet industrial expectations. The optimization can concern, for example, mechanical properties, increased material flexibility, increased rigidity, increased resilience, and improvement of water absorption capacity [bib_ref] Bionanocomposite films based on polysaccharides from banana peels, Oliveira [/bib_ref] [bib_ref] Frache, A. Plasticizers, antioxidants and reinforcement fillers from hazelnut skin and cocoa..., Battegazzore [/bib_ref].
## Bioplastics mechanical and physical properties
In order to assess the suitability of a biomaterial for a given sector and to establish the service life that can be expected, an evaluation of its mechanical-physical properties is mandatory. The main mechanical properties that are typically tested after the production of a bioplastic are the ultimate tensile strength, the Young's Modulus, and the elongation at break. The ultimate tensile strength, or just tensile strength, indicates the maximum stress that a material can withstand before fracturing, while the Young's Modulus, also known as elastic modulus, defines the stiffness of a material: the bigger is its value, the stiffer the material [bib_ref] Semichemical fibres of Leucaena collinsii reinforced polypropylene composites: Young's Modulus analysis and..., Granda [/bib_ref]. As regard to the elongation at break values, they are a measure of material ductility and depend on the rate (crosshead speed) and the temperature. The elongation at break value is, generally, very small and close to zero for brittle materials. On the contrary, materials with a better capacity to handle an excessive load without failure show higher elongation than 100% [bib_ref] Chemometrics and Intelligent Laboratory Systems Prediction of elongation at break for linear..., Palomba [/bib_ref]. Clearly, all these properties are affected by the chemical structure, the orientation degree of the polymers, and the crystallinity of the material, as well as by the eventual presence of fibers that act as reinforcement, or plasticizers [bib_ref] Study on Mechanical Properties of Natural-Glass Fibre Reinforced Polymer Hybrid Composites: A..., Sanjay [/bib_ref] [bib_ref] Food Bioscience The effect of plasticizers on the functional properties of biodegradable..., Suderman [/bib_ref]. Plasticizers are low volatile molecules, added to bio-polymeric materials to ensure an increasing of their extensibility, dispensability, flexibility, and elasticity [bib_ref] Natural-based plasticizers and biopolymer films: A review, Vieira [/bib_ref]. Several theories to explain the mechanisms of plasticization action have been proposed [bib_ref] Effects of hydrophilic plasticizers on mechanical, thermal, and surface properties of chitosan..., Suyatma [/bib_ref]. The lubrication theory states that plasticizers, by interspersing themselves, act as internal lubricants by reducing frictional forces between polymer chains. The gel theory, instead, postulates that the rigidity of polymers comes from three-dimensional structures, and plasticizers take effect by breaking polymer-polymer interactions (e.g., hydrogen bonds and van der Waals or ionic forces). The free volume theory states a plasticization as a study of ways to increase free volume and is useful in explaining the lowering of the glass transition temperature (T g ) by a plasticizer. Ideal plasticizers should be miscible and compatible in all proportions with plastic components, and they may be added to polymers in solution (dispersion technique) or after solvents have been removed (absorption technique) [bib_ref] Corn protein-based thermoplastic resins: Effect of some polar and amphiphilic plasticizers, Di Gioia [/bib_ref] [bib_ref] Tensile properties and water absorption of zein sheets plasticized with oleic and..., Santosa [/bib_ref]. Water, oligosaccharides, polyols, and lipids are different types of plasticizers widely used for edible films and coatings [bib_ref] Physical and mechanical properties of a new edible film made of pea..., Saberi [/bib_ref].
For hydrophilic polymers, polyols have been proven to be very efficient as plasticizers [bib_ref] Physical, mechanical, barrier, and thermal properties of polyol-plasticized biodegradable edible film made..., Ghasemlou [/bib_ref] [bib_ref] Water vapor and oxygen-barrier performance of corn-Zein coated polypropylene films, Tihminlioglu [/bib_ref]. In detail, for bio-based polymers obtained from fruits and vegetables waste, the recent researchers have focused on the usage of glycerol [bib_ref] Development of bio-based films and 3D objects from apple pomace, Gustafsson [/bib_ref] [bib_ref] Biodegradable plastic production from fruit waste material and its sustainable use for..., Yaradoddi [/bib_ref] [bib_ref] The Development of Banana Peel/Corn Starch Bioplastic Film: A Preliminary Study, Sultan [/bib_ref] [bib_ref] Effect of glycerol, nanoclay and graphene oxide on physicochemical properties of biodegradable..., Faradilla [/bib_ref] [bib_ref] Production of Pectin-Cellulose Biofilms: A New Approach for Citrus Waste Recycling, Bátori [/bib_ref] and sorbitol [bib_ref] Bioplastic from Jackfruit Seeds and Rice, Lestari [/bib_ref]. Glycerol content has significant effects on the mechanical properties as well as on the dynamic rheological behavior of thermo-molded bioplastics. Indeed, it was demonstrated that the increasing of glycerol content decreases tensile strength and Young's modulus but improves ductility at room temperature [bib_ref] Morphology and mechanical properties of thermo-molded bioplastics based on glycerol-plasticized wheat gliadins, Sun [/bib_ref]. Several studies on plasticization of chitosan films revealed that poly(ethylene glycol) (PEG) could improve the elastic properties of the chitosan biopolymer. Caner et al. [bib_ref] Chitosan film mechanical and permeation properties as affected by acid, plasticizer, and..., Caner [/bib_ref] observed that chitosan plasticization using PEG was stable until nine weeks of storage [fig_ref] Figure 3: Effects of the plasticizers' addition on mechanical and physical properties of bioplastic... [/fig_ref].
## Food waste as feedstock for bioplastic production
The most recent research concerning bioplastic production focuses on by-products and waste materials of food industries. According to the Food and Agriculture Organization (FAO) of the United Nations, every year an estimated 1.3 billion tons of food is wasted globally from all stages of the food supply chain including post-production, handling/storage, manufacturing, wholesale/retail, and consumption. Since food waste landfilling yields undesirable results, such as greenhouse gas (GHG) emissions and groundwater contamination, their valorization through bioplastics production could offer the possibility to overcome their disposal problem by renewable sustainable processes [bib_ref] Production of bioplastic through food waste valorization, Tsang [/bib_ref]. In addition to the mechanical properties, plasticizers also affect the physical properties of the biomaterials, which means water vapor permeability (WVP), oxygen permeability (OP), and water contact angle (WCA) [fig_ref] Figure 3: Effects of the plasticizers' addition on mechanical and physical properties of bioplastic... [/fig_ref] [bib_ref] Development of bioplastic materials: From rapeseed oil industry by products to added-value..., Delgado [/bib_ref]. These parameters serve as indicators of how easily water vapor or oxygen can penetrate a biodegradable material and they are a function of the hydrophilicity and hydrophobicity ratio of the main components by which the biomaterial is made. As to water contact angle, which is measured as the angle between the baseline of a drop deposited on the surface of the material and the tangent at the drop boundary, it increases with increasing surface hydrophobicity [bib_ref] Water vapor and oxygen-barrier performance of corn-Zein coated polypropylene films, Tihminlioglu [/bib_ref]. Since the surfaces degree of hydrophobicity is important to ensure good barrier properties, the evaluation of WVP, OP, and WCA is demanded. Recently, Aguilar et al. [bib_ref] Effect of different polyols as plasticizers in soy based bioplastics, Aguilar [/bib_ref] found that different physical and mechanical properties could be achieved at room temperature for bioplastics based on a soy protein isolated as a byproduct of the soy oil industry and added with different polyols, i.e., s (glycerol (GLY), ethylene glycol (EG), diethylene glycol (DEG) and triethylene glycol (TEG). In this sense, TEG-bioplastics were opaque, brittle, and also had a higher water uptake capacity, while EG-bioplastics were more ductile and translucent, absorbing much less water when immersed. Only GLY and TEG remained in the bioplastic after 9 days of storage at 50 - C, pointing out the volatility of EG and DEG causing a major ageing effect. On the other hand, it was also observed that sugars like sucrose and trehalose could act as plasticizers in presence of water. In detail, when water is included in the bioplastic formulation together with glycerol, sugars are solubilized within the aqueous fraction, and then play a plasticizer role in the bioplastics. In that case, lower viscoelastic properties and greater water absorption ability are generally detected [bib_ref] Evaluation of Composition on Processability and Water Absorption of Wheat Gluten-Based Bioplastics, Alonso-González [/bib_ref].
## Food waste as feedstock for bioplastic production
The most recent research concerning bioplastic production focuses on by-products and waste materials of food industries. According to the Food and Agriculture Organization (FAO) of the United Nations, every year an estimated 1.3 billion tons of food is wasted globally from all stages of the food supply chain including post-production, handling/storage, manufacturing, wholesale/retail, and consumption. Since food waste landfilling yields undesirable results, such as greenhouse gas (GHG) emissions and groundwater contamination, their valorization through bioplastics production could offer the possibility to overcome their disposal problem by renewable sustainable processes [bib_ref] Production of bioplastic through food waste valorization, Tsang [/bib_ref]. In addition, the production of value-added products while reducing the volume of waste is expected to reduce the production cost of biodegradable plastics, e.g. compared to conventional routes of production using overpriced pure substrates [bib_ref] Production of bioplastic through food waste valorization, Tsang [/bib_ref].
Food waste (FW) can be valorized in several ways in order to produce bioplastics [fig_ref] Figure 4: Figure 4 [/fig_ref].
## Food waste as feedstock for bioplastic production
The most recent research concerning bioplastic production focuses on by-products and waste materials of food industries. According to the Food and Agriculture Organization (FAO) of the United Nations, every year an estimated 1.3 billion tons of food is wasted globally from all stages of the food supply chain including post-production, handling/storage, manufacturing, wholesale/retail, and consumption. Since food waste landfilling yields undesirable results, such as greenhouse gas (GHG) emissions and groundwater contamination, their valorization through bioplastics production could offer the possibility to overcome their disposal problem by renewable sustainable processes [bib_ref] Production of bioplastic through food waste valorization, Tsang [/bib_ref]. In addition, the production of value-added products while reducing the volume of waste is expected to reduce the production cost of biodegradable plastics, e.g. compared to conventional routes of production using overpriced pure substrates [bib_ref] Production of bioplastic through food waste valorization, Tsang [/bib_ref].
Food waste (FW) can be valorized in several ways in order to produce bioplastics [fig_ref] Figure 4: Figure 4 [/fig_ref]. Conversion of food wastes into bioplastics could occur through biopolymers extraction and their mechanical manufacturing (extrusion, casting, molding, or combination of them). In more complex processes, food wastes are used as substrate for bacterial fermentation in order to produce biopolymers or as raw material for the extraction of other natural components, such as fibers, which act as reinforcing agents and/or natural filler of biocomposites. . In more complex processes, food wastes are used as substrate for bacterial fermentation in order to produce biopolymers or as raw material for the extraction of other natural components, such as fibers, which act as reinforcing agents and/or natural filler of biocomposites.
It is often used as substrate for bacterial fermentation to obtain natural polyesters, namely PHA and polylactic acid (PLA). When used to produce PHAs, food waste is a prime candidate for an inexpensive carbon source, due to its widespread availability and the potential to solve significant waste problems. In this case, physical, thermochemical, and biological pre-treatments of the FW are requested. Briefly, as reported by Tsang et al. [bib_ref] Production of bioplastic through food waste valorization, Tsang [/bib_ref] , a preliminary liberation of monomers from the FW (e.g., lignocellulosic components) with increasing accessibility of proteins, lipids, and polysaccharides (e.g., starch and cellulose), for subsequent enzymatic hydrolysis and fermentation, are essential. After the pre-treatment, the FW is ready for fermentation step in presence of bacteria, by using several cultivation strategies.
Other simple technologies for the production of bioplastics involve the direct extraction, from the food-waste-stream, of the biopolymeric components that are worked to give the finished products. More complex processes, on the other hand, require additional steps through which the biomass or the biopolymers extracted from it are used as reinforcements or fillers for the realization of biocomposites [bib_ref] Recent Developments in Biocomposites Reinforced with Natural Biofillers from Food Waste, Bashir [/bib_ref].
In many cases, the different "bioplastic formulations" need to be blended with additives in order to optimize some properties of the materials, such as thermal instability, high water vapor, brittleness, and low melt strength. Plasticizers, like glycerol, for example, are often required to improve the processability and the mechanical properties by interrupting hydrogen bonding and reducing the interactions between the biopolymers chains [bib_ref] Natural-based plasticizers and biopolymer films: A review, Vieira [/bib_ref].
However, both formulations, biopolymerics and biocomposites, are lastly processed to obtain biofilms or three-dimensional objects by applying conventional mechanical techniques: extrusion, molding, casting, or a combination of them [bib_ref] Development of bio-based films and 3D objects from apple pomace, Gustafsson [/bib_ref] [bib_ref] Bioplastic Production from Microalgae: A Review, Cinar [/bib_ref] [bib_ref] Orange peel-derived pectin jelly and corn starch-based biocomposite film with layered silicates, Çokaygil [/bib_ref]. All these processing methods selected for the manufacture of food waste-based bioplastics play an important role in their final properties [bib_ref] Effect of processing on the viscoelastic, tensile and optical properties of albumen/starch-based..., González-Gutiérrez [/bib_ref]. Extrusion is a highly efficient way for the continuous shaping of biomaterials, and it consists in pushing the bioplastic dough against an orifice with the desired geometry and dimensions. The mass of the dough inside the extrusion chamber is compacted, and the semi-finished product that comes out is cut to give the wanted length. Instead, with casting and molding, the dough is respectively poured or pressed against a rigid frame.
Compression molding technique has been widely employed for the development of biofilms or 3D objects without the use of any solvent or binder [bib_ref] Development of bio-based films and 3D objects from apple pomace, Gustafsson [/bib_ref] [bib_ref] A Solvent-Free Approach for Production of Films from Pectin and Fungal Biomass, Gurram [/bib_ref]. With this technique, the waste of interest or its dried extract is finely powdered and subjected to high temperatures and pressures through a heated press. Under this thermo-mechanical treatment, proteins undergo denaturation and dissociation leading to the formation of new links and their aggregation to new forms; in addition, biopolymers show a self-binding ability that is exploited to produce three-dimensional objects. Furthermore, the molding method is more suitable for industrial applications since it is characterized by lower energy demand and processing time compared to other techniques like solution casting [bib_ref] A Solvent-Free Approach for Production of Films from Pectin and Fungal Biomass, Gurram [/bib_ref].
In the last 10 years, several food wastes have been used as raw material for the production of bioplastics, including biocomposites; mostly fruit and vegetable wastes rich in polysaccharides (such as cellulose, starch, pectin) and in fibers [bib_ref] The Development of Banana Peel/Corn Starch Bioplastic Film: A Preliminary Study, Sultan [/bib_ref] [bib_ref] Bioplastic from Jackfruit Seeds and Rice, Lestari [/bib_ref] [bib_ref] Effect of processing on the viscoelastic, tensile and optical properties of albumen/starch-based..., González-Gutiérrez [/bib_ref] [bib_ref] Mechanical properties of bioplastic form cellulose nanocrystal (CNC) mangosteen peel using glycerol..., Muhammad [/bib_ref] [bib_ref] Optimization of pectin extraction from banana peels with citric acid by using..., Oliveira [/bib_ref] [bib_ref] Recycled wastes of tomato and hemp fibres for biodegradable pots: Physico-chemical characterization..., Schettini [/bib_ref] [fig_ref] Table A1: Cont. [/fig_ref]. In the next paragraphs, a focus on the main uses of fruits and vegetables wastes for the production of bioplastics will be provided.
## Biopolymers-based plastics
Biopolymers extracted from fruits and vegetables wastes show different characteristics and properties that make them more or less suitable for the production of eco-friendly materials . The extraction of biopolymers from food waste could be achieved chemically or enzymatically. Enzymatic processes are widely considered "clean" since they are solvent-free [bib_ref] Biobased technologies for the efficient extraction of biopolymers from waste biomass, Jha [/bib_ref] ; however, this technology is still hindered by economic and technical limits, i.e., costly enzymes and long processing period. Because of the high cost and time-consuming nature, the production of bioplastics through sugar's bacterial fermentation, occurring in agricultural waste, is disadvantageous. Therefore, chemical extraction with solvents could be considered as the best solution at the lowest amount of energy. . Properties associated with the main biopolymers extracted from fruits and vegetable wastes for bioplastics production.
## Biopolymer name biopolymer type properties
## Fruits and vegetable wastes used as biopolymer source
Cellulose Polysaccharide
Highly structured intermolecular hydrogen bonding network; impossibility of melting or dissolution by standard processes such as thermoforming.
Banana peels, carrots waste, cauliflower waste, cocoa pod husks, orange peels, parsley steams, radicchio waste, rice hulls, spinach steams, tea leaves waste.
## Starch polysaccharide
Strong inter-and intra-molecular hydrogen bonding; water sensitivity and poor fowability; brittleness.
Banana peels, cassava peels, potato peels.
## Pectin polysaccharide
Gelling ability but poor tensile and barrier properties; water sensitivity.
Apple pomace, banana peels, citrus waste, orange peels
## Cutin
## Polyester of hydroxy fatty acids
Amorphous and flexible three-dimensional polymer; hydrophobic, low water sensitivity.
## Tomato waste
One of the main macromolecules extracted from fruits and vegetables waste and used for the production of biomaterials is the cellulose. The preparation of the pure cellulose bioplastics from bio-sources is not easy due to the highly structured intermolecular hydrogen bonding network of the polymer, which cannot be melted or dissolved by standard processes such as thermoforming [bib_ref] Direct transformation of edible vegetable waste into bioplastics, Bayer [/bib_ref] [bib_ref] Understanding Multiscale Structural Changes during Dilute Acid Pretreatment of Switchgrass and Poplar, Pingali [/bib_ref]. Thus, the cellulose is usually used in industrial applications in the form of derivatives, such as esters or ethers, from which cellulose is then regenerated [bib_ref] Recent advances in regenerated cellulose materials, Wang [/bib_ref]. Nevertheless, in recent years, several biomaterials have been manufactured through amorphous cellulose extracted from vegetables by using different solvents. Bayer et al. [bib_ref] Direct transformation of edible vegetable waste into bioplastics, Bayer [/bib_ref] obtained amorphous cellulose-based biomaterial by digesting parsley and spinach stems, rice hulls, and cocoa pod husks wastes in trifluroacetic acid (TFA), followed by casting and evaporation. TFA is a naturally occurring and biodegradable organic acid that can co-solubilize cellulose with other contained organic matter; it breaks the hydrogen bonds between neighboring cellulose chains (intersheet hydrogen bonds) and partial trifluoroacetylates OH groups of cellulose with formation of amorphous materials [bib_ref] Inverse Temperature-Dependent Pathway of Cellulose Decrystallization in Trifluoroacetic Acid, Zhao [/bib_ref]. The mechanical properties of the produced cellulose-based biofilms were proved to be largely dependent on the starting biowaste. Indeed, cocoa pod husks biofilm displayed a tensile stress at break of approximately 30 MPa; whereas for the rice, the parsley, and the spinach-derived films, the obtained values were, respectively, 7 MPa, 5 MPa, and approximately 1 MPa, i.e., values close to elastomers and low density polyethylene thermoplastic [bib_ref] Direct transformation of edible vegetable waste into bioplastics, Bayer [/bib_ref]. Such higher stresses at break and strains for cocoa pod husks derived biomaterial were due to their significant number of triglycerides, i.e.oligomeric esters precursors of biopolymers. Instead, residual silica in the rice hulls derived material conferred a higher rigidity compared to parsley-and spinach-based biomaterials. UTS (ultimate tensile strength) at high Young's modulus comparable to poly(ethylene terephthalate) of bioplastics from cocoa pod husk could be compared with petroleum-based thermoplastics, such as high-density polyethylene and polypropylene. Rice straw was used also by Bilo et al. [bib_ref] A sustainable bioplastic obtained from rice straw, Bilo [/bib_ref] to produce a new cellulose-based bioplastic material through a process that involved the digestion with TFA, preceded by an extraction pre-treatment performed in a rapid dynamic solid-liquid extractor. With this process, a bio-material with better mechanical properties, compared to those evaluated by Bayer et al. [bib_ref] Direct transformation of edible vegetable waste into bioplastics, Bayer [/bib_ref] , was obtained. Indeed, the tensile test of dried and wet dumbbell specimens allowed to ascertain tensile strengths and elongations at break equal to 45 MPa and 6.1% and 10 MPa and 63%, respectively [fig_ref] Table A1: Cont. [/fig_ref]. The replacement of TFA with a diluted aqueous cloridric acid (HCl) solution has been found to be a better method to obtain biofilm evidencing higher stiffness and lower ductility. Perotto et al. [bib_ref] Bioplastics from vegetable waste: Via an eco-friendly water-based process, Perotto [/bib_ref] used this water-based process to convert carrot, parsley, radicchio, and cauliflower wastes into flexible bioplastic films made by cellulose crystals fused together, with some soluble components like pectin and sugars blended homogeneously acting as plasticizers. Compared to the oil-based polymers, the Young's Modulus (1.3 ± 0.2 GPa) and the UTS (38 ± 5 GPa) of the carrot bioplastics are similar to those of polypropylene, albeit with lower elongation. The mild conditions of the conversion process were demonstrated also to preserve the functional properties of the original vegetable, like the anti-oxidant activity [bib_ref] Bioplastics from vegetable waste: Via an eco-friendly water-based process, Perotto [/bib_ref]. An aqueous HCl solution was used also by Yaradoddi et al. [bib_ref] Biodegradable plastic production from fruit waste material and its sustainable use for..., Yaradoddi [/bib_ref] to produce a cellulose-based biofilm from banana peels; anyway, in this case, no mechanical tests were conducted in order to evaluate the strength of the material.
Although TFA and HCl are efficient acids for vegetable waste derived-cellulose dissolution, their utilization, and waste production remain problematic if considering the principles of green chemistry. Recently, a less harmful acid, i.e., citric acid, has been used by Liu et al. [bib_ref] Green and facile preparation of hydrophobic bioplastics from tea waste, Liu [/bib_ref] in a green, non-toxic, waste-free method of synthesizing hydrophobic bioplastic films from spent tea leaves. The resultant material exhibited an ultimate tensile strength of 6.16 MPa and an elongation at break of 13.33%, thus it had a lower stiffness compared to oil-derived polymers, such as PP [fig_ref] Table A1: Cont. [/fig_ref]. Since citric acid was found to not fully react with the tea waste matrix, the authors hypothesized that unreacted citric acid acted as a hygroscopic plasticizer in the bioplastic films.
In addition to cellulose, starch, i.e., a polymer consisting of a long chain of two glucose units joined together, namely branched polymerized amylopectin and amylose, can be considered as an effective eco-solution for the production of biomaterials, because it is inexpensive and easily available . Starch for the production of biofilm have been obtain from different sources, principally potatoes, banana and cassava peels [bib_ref] The Development of Banana Peel/Corn Starch Bioplastic Film: A Preliminary Study, Sultan [/bib_ref] [bib_ref] Bioplastic Synthesis Using Banana Peels And Potato Starch And Characterization, Beevi [/bib_ref] [bib_ref] Production of bioplastic from potato peel waste and investigation of its biodegradability, Arikan [/bib_ref] [bib_ref] The effect of kaffir lime (Citrus hystrix DC) essential oil on bioplastic..., Masruri [/bib_ref] [bib_ref] Food packaging development of bioplastic from basic waste of cassava peel (manihot..., Dasumiati; Saridewi [/bib_ref] [bib_ref] Characterization of bioplastic based from cassava crisp home industrial waste incorporated with..., Fathanah [/bib_ref] [bib_ref] Bioplastics production from agricultural crop residues, Samer [/bib_ref]. Arikan et al. [bib_ref] Production of bioplastic from potato peel waste and investigation of its biodegradability, Arikan [/bib_ref] investigated the production of bioplastics from potato peels waste, obtaining satisfactory results in terms of biodegradability (the time requested for the complete biodegradation of the material was 28 days, see [fig_ref] Table A1: Cont. [/fig_ref]. However, native starch-based films are limited to high water affinity and brittleness, therefore other natural biopolymers are often added as fillers to modify and improve films' properties. As example, Dasumiati et al. [bib_ref] Food packaging development of bioplastic from basic waste of cassava peel (manihot..., Dasumiati; Saridewi [/bib_ref] and Fathanah et al. [bib_ref] Characterization of bioplastic based from cassava crisp home industrial waste incorporated with..., Fathanah [/bib_ref] improved the mechanical properties of cassava peels derived starch by introducing chitosan as filler. In another work, proteins derived from soybeans waste were mixed with starch and glycerol as plasticizer, since proteins structure consists of stable three-dimensional networks which do not ensure material with enough plasticity [bib_ref] Development of bio based plastic materials for packaging from soybeans waste, Muhammad [/bib_ref]. Instead, Sultan et al. [bib_ref] The Development of Banana Peel/Corn Starch Bioplastic Film: A Preliminary Study, Sultan [/bib_ref] developed bioplastic film from a combination of banana peels derived starch and different concentrations of corn starch (1% up to 5%) as co-biopolymer. Based on the results obtained, the film with 4% of corn starch gave the highest tensile strength 34.72 N/m 2 compared to other samples, while the authors stated that the biofilms with 3% of corn starch were resistant to water uptake by absorbing water up to 60.65% [fig_ref] Table A1: Cont. [/fig_ref]. However, it should be considered that this value is considerably higher compared to conventional plastics such as PP, whose percentage of water absorption after 24 h of immersion ranges between 0.01 and 0.03.
Besides being a starch source, banana peels have been shown also to contain a good percentage of pectins [bib_ref] Dietary fibre components and pectin chemical features of peels during ripening in..., Emaga [/bib_ref]. Pectins are a family of covalently linked galacturonic acid-rich plant cell wall polysaccharides with functions in plant growth, morphology, and development; they also serves as gelling and stabilizing polymers in diverse foods [bib_ref] Pectin structure and biosynthesis, Mohnen [/bib_ref]. The production of pectin-based biofilms typically involves the introduction of cellulose and hemicellulose components, since polysaccharidic films show poor tensile and barrier properties compared to those of petroleum-derived polymers. To this regard, Oliveira et al. [bib_ref] Bionanocomposite films based on polysaccharides from banana peels, Oliveira [/bib_ref] isolated pectin from banana peels in order to prepare a biofilm whose tensile strength was increased through the addition of cellulose nanocrystals (CNCs) extracted from the same banana wastes (tensile strength values obtained were about 7 MPa, see [fig_ref] Table A1: Cont. [/fig_ref]. The tensile strength increase was due to favorable nanocrystal-pectin interactions as well as to the reinforcing effect through stress transfer at the nanocrystal-pectin interface [bib_ref] Mechanical and barrier properties of nanocrystalline cellulose reinforced chitosan based nanocomposite films, Khan [/bib_ref].
Aside from the poor mechanical properties, the strong hydrophilic character of polysaccharidic films makes them dissolve in contact with water, limiting their applications [bib_ref] Bionanocomposite films based on polysaccharides from banana peels, Oliveira [/bib_ref]. To overcome the high water permeability, citric acid could be added, as it crosslinks polysaccharide films by forming covalent diester linkages between two of their carboxyl groups and hydroxyl groups of different polysaccharide chains [bib_ref] Influence of citric acid and curing on moisture sorption, diffusion and permeability..., Olsson [/bib_ref]. In the previous mentioned work reported by Oliveira et al. [bib_ref] Bionanocomposite films based on polysaccharides from banana peels, Oliveira [/bib_ref] , the presence of citric acid was ascertained to decrease the water vapor permeability from 3.31 to 3.10 g·mm·kPa −1 ·h −1 ·m −2 . Citric acid was used also for the processing of orange and apple wastes (OW and AW) in order to obtain a biodegradable material through a casting method in which cellulose and hemicelluloses were suspended in the pectin solution and further dried to a film [bib_ref] Development of bio-based films and 3D objects from apple pomace, Gustafsson [/bib_ref] [bib_ref] Production of Pectin-Cellulose Biofilms: A New Approach for Citrus Waste Recycling, Bátori [/bib_ref]. In detail, Batori et al. [bib_ref] Production of Pectin-Cellulose Biofilms: A New Approach for Citrus Waste Recycling, Bátori [/bib_ref] used a solution of citric acid and glycerol to form a biofilm from OW, exploiting the gelling ability of pectin and the strength of its cellulosic fibers. The tensile strengths of the films were 31.67 ± 4.21 and 34.76 ± 2.64 MPa, respectively, for the oven-dried and incubator-dried films. These values were within the range of different commodity plastics. In addition, anaerobic digestion was performed for testing the biodegradability of the material and a time of 15 day was requested to reach 90% of degradation. Instead, from a mixture of apple pomace waste (AW) and glycerol, a fluffier and connected structure (tensile strength 3.27 ± 0.31 MPa without including a washing step) was obtained by Gustaffson et al. [bib_ref] Development of bio-based films and 3D objects from apple pomace, Gustafsson [/bib_ref] , but with significant flexibility, similar to those of PP (elongation %: 55.41 ± 5.38, [fig_ref] Table A1: Cont. [/fig_ref].
The same authors [bib_ref] Development of bio-based films and 3D objects from apple pomace, Gustafsson [/bib_ref] made an attempt to produce bioplastics by using solvent-free mechanical processing of AW. Compression molding technique has been widely employed for the development of pectin-based biofilm or 3D objects without the use of any solvent or binder [bib_ref] Development of bio-based films and 3D objects from apple pomace, Gustafsson [/bib_ref] [bib_ref] A Solvent-Free Approach for Production of Films from Pectin and Fungal Biomass, Gurram [/bib_ref]. Gurram et al. [bib_ref] A Solvent-Free Approach for Production of Films from Pectin and Fungal Biomass, Gurram [/bib_ref] applied a compression molding method for production of bioplastic films from citrus peel derived pectin. Moreover, free sugars and water-soluble nutrients were extracted from citrus waste and employed for cultivation of the filamentous fungus Rhizopus oryzae, whose biomass was incorporated into the pectin films. The addition of fungal biomass (up to 20%) enhanced the tensile strength (16.1-19.3 MPa) and reduced the water vapor permeability of the pectin films [fig_ref] Table A1: Cont. [/fig_ref].
In addition to the cellulose, starch, and pectine, a sustainable melt polycondensation of unsaturated and polyhydroxylated fatty acids recovered from tomato pomace agro-wastes, has been recently carried out in order to obtain an aliphatic polyester type of bioplastic without the use of solvents during the reaction [bib_ref] Sustainable polycondensation of multifunctional fatty acids from tomato pomace agro-waste catalyzed by..., Heredia-Guerrero [/bib_ref]. Polyhydroxylated fatty acids are found in tomato pomace in the form of cutin, i.e., a biopolyester mainly composed of C [bib_ref] Comparison of the aerobic biodegradation of biopolymers and the corresponding bioplastics: A..., Polman [/bib_ref] and C 18 fatty acids monomers linked together and forming an amorphous and flexible three-dimensional polymer matrix [bib_ref] Plant cutin genesis: Unanswered questions, Domínguez [/bib_ref]. Since cutin isolation to produce bioplastics is a long multistep process and unsuitable for large-scale applications, a direct depolymerized of tomato pomace through alkaline hydrolysis, followed by monomers polycondensation, has been proposed by Heredia-Guerrero et al. [bib_ref] Sustainable polycondensation of multifunctional fatty acids from tomato pomace agro-waste catalyzed by..., Heredia-Guerrero [/bib_ref] as a simpler and cheaper alternative. To that purpose, the influence of different temperatures, reaction times, and amounts of tin (II) 2-ethylhexanoate used as a catalyst, was evaluated. Synthesized tomato pomace bioplastics showed an amorphous molecular structure, whose mechanical properties were dependent on the degree of polymerization. In detail, an increase in hardness of the polyesters synthesized at higher reaction temperatures and amount of catalyst was detected (~1.8 MPa for biopolymers obtained at 125 - C and 0 mmol of catalyst against~26.3 MPa for biopolymers obtained at 175 - C and 0.1 mmol of catalyst), since in those conditions a higher degree of polymerization was achieved. The water-contact angles of more polymerized samples were around 109 - , which are values comparable to traditional hydrophobic polymers such as PDMS and PTFE. Concerning water uptakes, the obtained percentages were typical of low-absorbing plastics (2.1-61%).
## Fruits and vegetables waste usage for biocomposites production
More often, biopolymers extracted from fruits and vegetable wastes are blended with other polymers whose mechanical and physical properties are not suitable to accomplish commercially acceptable products [bib_ref] Orange peel-derived pectin jelly and corn starch-based biocomposite film with layered silicates, Çokaygil [/bib_ref] , thus realizing composite materials known as biocomposites.
Biocomposite materials are usually made by a polymeric matrix coming from a renewable and available origin, such as polysaccharides, reinforced by natural fillers. Examples of natural fillers are layered silicates. They can be synthetized from silica naturally occurring in leaves, husks, blades, hulls, roots, and stems of many terrestrial and marine plants, including wheat, rice, horsetails, oats, barley, grasses, and algae. Among bio-wastes, one of the most silica-rich sources is rice husks, which is largely available, typically 20-22 wt% of rice grains. It is been used by Deng et al. [bib_ref] Simple process for synthesis of layered sodium silicates using rice husk ash..., Deng [/bib_ref] for layered silicates synthesis. Layered silicates (LSs) have hydrophilic characteristics owing to the presence of inorganic cations (Na + and Ca 2+ ) in the interlayer spacing; hence, they are miscible with different hydrophilic polymers, including starch and pectin, able to compensate their rheological property differences [bib_ref] Orange peel-derived pectin jelly and corn starch-based biocomposite film with layered silicates, Çokaygil [/bib_ref].
Despite not being recovered from vegetables wastes, Cokaygil et al. [bib_ref] Orange peel-derived pectin jelly and corn starch-based biocomposite film with layered silicates, Çokaygil [/bib_ref] used LSs as natural filler to prepare biocomposite films having corn starch and pectin extracted from orange peels as a polymeric matrix. Different pectin jelly-to-starch weight ratios (63/37, 60/40, 57/43, and 54/46 w/w) were considered when formulating the film ingredients. Furthermore, to enhance the compatibility and wettability among starch, LS, and pectin, starch and LSs were chemically modified through reaction with propylene oxide and hexadecyltrimethylammonium chloride, respectively. Among all the films considered, pectin jelly/modified starch-based biocomposite film (54/46 w/w) containing 0.25 wt % of LSs was found to be the most promising in terms of texture structure and mechanical integrity.
In the most recent years, wastes of agro-food industries have attracted attention also as sources of natural fibers exploitable as reinforcing elements of biodegradable biocomposite materials. Bio fibers, which are natural polymers, could be obtained from a large variety of fruits and vegetables [bib_ref] Biofibres, biodegradable polymers and biocomposites: An overview, Mohanty [/bib_ref] , thus reflecting several characteristic properties unlike conventional fibers. Undoubtedly, conventional fibers for instance glass, carbon, and aramid can be produced with a definite range of properties, with a higher cost as well. In 2013, Schettini et al. [bib_ref] Recycled wastes of tomato and hemp fibres for biodegradable pots: Physico-chemical characterization..., Schettini [/bib_ref] developed a novel biocomposite by using hemp and tomato peels and seeds fibers as natural reinforcement for sodium alginate polymer, in order to produce biodegradable pots in agriculture. Three different compositions of biocomposites were prepared by varying the percentage of tomato and hemp fibers added to sodium alginate water solution. By soaking the doughs with a calcium chloride solution, a three-dimensional and stable crosslinked network of calcium alginate was obtained as well and it was subjected to investigation of its functionality, physico-chemical and mechanical behavior. As reported by the authors, by increasing the hemp fibers content, a general enhancement of the mechanical parameters of both un-crosslinked and crosslinked samples was registered, since fibers from hemp strands are more rigid, stiff, and long in comparison to the more flexible and short fibers from tomato peels and seeds [bib_ref] Bio-based composites from waste agricultural residues, Ashori [/bib_ref]. Moreover, crosslinked biocomposites showed a lower rigidity and strength with respect to their corresponding un-crosslinked counterparts (Young Modulus for un-crosslinked 100% tomato fibers biocomposite was 63.62 MPa; while for crosslinked 100% tomato fibers biocomposite Young Modulus was 48.05 MPa). Such a behavior was due to the loss of adhesive properties, which occurs when carboxylated and hydroxyl groups of alginate are strongly engaged in physical interaction with calcium ions during the crosslinking process, thus reducing the bonding strength between the matrix and the fibers [bib_ref] Effect of cross-linking with calcium ions on the physical properties of alginate..., Russo [/bib_ref]. However, these obtained values were all comparable to those of conventional plastics. Instead, Mathivanan et al. [bib_ref] The study of mechanical properties of pineapple leaf fibre reinforced tapioca based..., Mathivanan [/bib_ref] used different percentages of pineapple leaf fibers to reinforce tapioca based bioplastic resin through a method based on extrusion followed by hot compression molding. The 30% composition showed the best average modulus value among other composition, leading to the conclusion that the increase of pineapple leaf fibers increases the modulus strength of the composite.
Since passion fruit waste contains about 60% of fibers [bib_ref] Dietary fiber components in yellow passion fruit rind-A potential fiber source, Yapo [/bib_ref] that, when dried, could be used as reinforcement of thermoplastic starch, Moro et al. [bib_ref] Bioplastics of Native Starches Reinforced with Passion Fruit Peel, Moro [/bib_ref] tried to develop an extruded starchy bioplastic, reinforced with different content of passion fruit peel (0, 4, 10, 16, and 20%), glycerol and starch mix, recovered from corn and cassava. In this way, it was possible to obtain starch-based bioplastic with stronger and midterm elastic property (Tensile strength ranged between 1.6 MPa and 9.0 MPa, while the elongation at break values were between 24.7% and 54.5%, see [fig_ref] Table A1: Cont. [/fig_ref]. Despite this, the tensile strength values were lower of oil-derived polymers.
On the other hand, bio-blend of poly-butylene succinate (PBS) and poly-butyleneadipate-co-terephthalate (PBAT) has been recently proved to be strengthened in terms of higher modulus (3.0 GPa) and lower water absorption (3.4%) with the addition of Miscanthus fiber and oat hull followed by reactive extrusion of the dough [bib_ref] Sustainable green composites from biodegradable plastics blend and natural fibre with balanced..., Wu [/bib_ref]. Indeed, PBS alone has a tensile strength of around 26.5 MPa, elongation of 21.5%, and a modulus of 48 MPa. The incorporation of fiber or cellulose remarkably improves the Young's modulus of neat PBS.
For their rich content in lipids, lignin, and fibrous polysaccharide components (cellulose, hemicellulose), peanut hulls and cocoa shell waste (CW) and hazelnut skin (HS) extracts have also been introduced into synthetic elastomers matrices as reinforcement fillers and plasticizers [bib_ref] Frache, A. Plasticizers, antioxidants and reinforcement fillers from hazelnut skin and cocoa..., Battegazzore [/bib_ref] [bib_ref] DIY Bioplastics from Peanut Hulls Waste in a Starch-Milk Based Matrix, Troiano [/bib_ref] [bib_ref] Bioelastomers Based on Cocoa Shell Waste with Antioxidant Ability, Tran [/bib_ref]. Battegazzore et al. [bib_ref] Frache, A. Plasticizers, antioxidants and reinforcement fillers from hazelnut skin and cocoa..., Battegazzore [/bib_ref] made selective and serial extractions from CW and HS to recover bio-components for producing high-added value PLA and PP plastics. Briefly, a first extraction with diethyl ether mainly separated lipids, phospholipids, and triglycerides, which were worked as plasticizers. In the second extracted fractions, instead, phenolic compounds and flavonoids, such as gallic acid and catechin, were distinguished by UV spectroscopy; therefore, those fractions served as antioxidant and photo-stabilizer for PP. In addition, they positively influenced the PP thermal stability in air; indeed, the temperature of its maximum weight loss was increased from 319 - C to 330 - C and 345 - C by adding HS and CW extracts, respectively. Finally, the last fractions extracted acted as reinforcement filler for PLA and PP; their content linearly influenced the oxygen permeability of the obtained biomaterials [fig_ref] Table A1: Cont. [/fig_ref]. Instead, Tran et al. [bib_ref] Bioelastomers Based on Cocoa Shell Waste with Antioxidant Ability, Tran [/bib_ref] introduced cocoa shell waste powder within an acetoxy-poly(dimethylsiloxane) silicone network through a process that involved a physical mixing with a nontoxic solvent and casting into a mold, with the advantage of direct utilization of CW without any extraction or purification steps. In this case, the antioxidant activity of the final cross-linked bioelastomers was investigated, demonstrating very effective radical scavenging activity against 2,2-diphenyl-1-picrylhydrazyl free radical and 2,2 -azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation.
As PLA reinforcement, cellulose extracted from pumpkins peels, and subsequently acetylated, has also been used [bib_ref] Sustainable materials based on cellulose from food sector agro-wastes, Côto [/bib_ref]. In this case, the addition of 10% of acetylated cellulose enhanced the PLA's mechanical properties with an increase of the storage modulus at 40 - C of around 40%. More generally, cellulose or cellulose nanocrystals have been obtained from various vegetable or fruit waste, such as banana peels, pine flowers waste, rice straw, palm empty fruit bunch, sago waste, mangosteen peels, and also successfully employed as reinforcements of biopolymers, mainly starch [bib_ref] Mechanical properties of bioplastic form cellulose nanocrystal (CNC) mangosteen peel using glycerol..., Muhammad [/bib_ref] [bib_ref] Preparation of Nanocellulose Bioplastic with a Gradation Color of Red and Yellow, Nasihin [/bib_ref] [bib_ref] Mechanical and Morphology Studies of Bioplastic-Based Banana Peels, Azieyanti [/bib_ref] [bib_ref] The effect of agricultural waste nanocellulose on the properties of bioplastic for..., Iriani [/bib_ref] [bib_ref] Effect of cellulose fiber loading on the properties of starch-based films, Yacob [/bib_ref]. In addition, banana pseudostems waste has been recently used to isolate nanocellulose employed for the production of green composites enriched with nano-fillers, such as graphene oxide and nanoclay, and glycerol as plasticizer [bib_ref] Effect of glycerol, nanoclay and graphene oxide on physicochemical properties of biodegradable..., Faradilla [/bib_ref]. As regard to rice straw, besides being considered as reinforcement, its fibers were proved to act as flame-retardant fillers in combination with PLA and lignin by Dahy et al.. Typically, flame retardant used to reduce combustibility of the polymers, are halogen-based additives that act in the vapor phase by a radical mechanism to interrupt the exothermic processes, interfering with the combustion process during heating, pyrolysis, ignition, or flame spread. Instead, a more environmentally friendly alternative that contemplates the incorporation of natural fillers, like rice straw derived fibers, mainly acts to dilute the polymer and reduce the concentration of decomposition gases [bib_ref] Recent developments in the chemistry of halogen-free flame retardant polymers, Lu [/bib_ref].
## Environmental impacts of agro-food waste based bioplastics production
Nowadays, fruit and vegetable valorization is one of the main pillars of the circular economy; their use to substitute fossil resources for the production of plastics, is a widely accepted strategy towards sustainable development. In fact, the displacing of conventional plastics with food waste-based bioplastics can lead to considerable energy and GHGs emissions savings [bib_ref] Production of bioplastic through food waste valorization, Tsang [/bib_ref]. However, it should be noted that this is not always true. Further details about advantages and drawbacks related to the bioplastics use and production are given in . Despite being promoted as a safer alternative to their oil-based counterparts, bioplastics production involves major drawbacks. Indeed, bioplastics are generally not cost-competitive compared to conventional plastics and their production is plagued by low yields and being expensive. Moreover, some bioplastics have a shorter lifetime than oil-based plastics due to weaker mechanical and physical properties, such as greater water vapor permeability than standard plastic, being easy to tear like tissue paper, or being very brittle. Being compostable and biodegradable sounds great, but many bioplastics must follow a specific disposal procedure and require industrial composting in order to avoid being incinerated or going to landfill. On the other hand, biodegradable polymers require a controlled fate to kickstart the expected biodegradation process and as a result, it is nearly impossible to control and ensure the complete degradation of even potentially degradable plastic materials. Subsequently, when they are disposed of in an uncontrolled fashion, they will accumulate in the environment and fragment into microplastics (MPs). These MPs have proven to display diverse impacts over ingested organisms and ecosystem similar to those of conventional MPs, thus bioplastics could be a solution to MPs only if properly disposed of [bib_ref] Bioplastics: Missing link in the era of Microplastics, Shruti [/bib_ref]. . Main advantages and drawbacks related to the production and the use of bioplastic materials.
## Advantages drawbacks
## Production
Reduction of greenhouse gas emission; saving fossil fuels, possibility of using a local resource, less energy during the manufacturing cycle.
Use of croplands to produce items, not cost-competitive compared to conventional plastics
## Use
No toxic, no release of chemicals into food if used as packaging
Often characterized by thermal instability, brittleness, low melt strength, high water vapor and oxygen permeability; when hydrophilic polymers are used, they possess low water vapor barrier and vulnerability to degradation.
## Disposal
Biodegradable; broken down by naturally occurring bacteria; do not persist for many years in the environment.
Controlled fate in order to kickstart the expected biodegradation process; a specific disposal procedure must be followed to avoid they fragment into microplastics which accumulate in the environment.
As a matter of fact, the employment of fruits and vegetables waste as reinforcement of non-biodegradable polymers in drop-ins significantly increases the energy demand and CO 2 emission compared to biodegradable bioplastics [bib_ref] Benchmarking Bioplastics: A Natural Step Towards a Sustainable Future, Bhagwat [/bib_ref].
Therefore, when the aim is the production of new bioplastic materials from agro-food waste, the effective sustainability of the process should be evaluated. The sustainability of bio-based plastics production depends on several factors that are often summarized in the life cycle assessments (LCAs) of the products [bib_ref] Sustainability assessments of bio-based polymers, Hottle [/bib_ref]. Among them, there are availability of commercially viable quantities of renewable feedstock and agricultural waste, scalable and green production routes, cost and competition with synthetic polymers, and useful life and biodegradation/end of life treatment [bib_ref] Production of sustainable and biodegradable polymers from agricultural waste, Maraveas [/bib_ref]. Many of these aspects are very often not sufficiently deepened, thus making it difficult to assess environmental impacts associated with the agro-food waste-based bioplastics production.
The greenhouse gas emissions generated by food waste globally represent the third largest emitter in the world, thus any measure to reduce food waste, even to a small extent, may have a significant impact on overall environmental footprint [bib_ref] Don't scrap the waste: The need for broader system boundaries in bioplastic..., Kakadellis [/bib_ref]. However, even though the number of fruits and vegetable wasted every year are estimated to be around 484 million, the volume of waste produced does not predict the availability of agricultural waste for conversion into biomaterials. Indeed, a large quantity is employed in other competing applications such as bio-fertilizer and biogas production. In addition, not all the routes proposed for obtaining bio-materials are applicable on a large scale, since sometimes they require extensive and advanced processing. This mainly concerns biocomposites production, which is often based on obtaining fillers and reinforcements, such as cellulose nanocrystals, through complex treatment of the agro-waste [bib_ref] Mechanical properties of bioplastic form cellulose nanocrystal (CNC) mangosteen peel using glycerol..., Muhammad [/bib_ref] [bib_ref] Mechanical and barrier properties of nanocrystalline cellulose reinforced chitosan based nanocomposite films, Khan [/bib_ref]. On the contrary, more feasible and scalable processes allow bioplastics production after chemical extraction of agro-polymers from the food waste stream. A low environmental impact is associated with this step, as no harsh chemicals, like pyridine and diethyl ether, are used for the production of PHA, and potential occupational hazards are covered.
Regarding the end of life of the agro-food waste = based bioplastics, reuse and recycling are preferred solutions to energy recovery or disposal. However, to date, for materials other than bio-PE or bio-PET, there is no recycling stream established yet. An alternative is their composting, i.e., their aerobic biodegradation under controlled conditions of temperature, humidity, and aeration [bib_ref] Compostability of bioplastic packaging materials: An overview, Kale [/bib_ref]. Compostability is a clear benefit of agro waste-based bioplastics compared to conventional plastics, resulting in the creation of more valuable compost.
## Bioplastics market
Currently, the number of bioplastics produced annually in all the world represents only about one percent of the 360 million tons of plastic materials produced globally. However, due to the growing sensitivity towards the adoption of a "green and circular economy" dependent policy, the global bioplastics production capacity is set to increase from around 2.11 million tons in 2019 to approximately 2.43 million tons in 2024.
With a view to regional capacity development, Asia remains a major production hub with over 50 percent of bioplastics currently being produced there. Presently, only one-fifth of the production capacity is located in Europe. This share is predicted to grow to up to 27 percent by 2023. The expected growth will be supported by recently adopted policies in several European Member States, such as Italy and France.
Innovative biopolymers, such as bio-based PP, bio-based PET, bio-based PA, and PHAs continue to drive the growth in bioplastic production. To date, they make up for 40 percent (0.8 million tons) of the global bioplastics production capacities. Bioplastics materials are currently used in an increasing number of markets: From packaging, catering products, consumer electronics, automotive, agriculture/horticulture, and toys to textiles. Among these several market segments, electronics is the less developed (only about 2% of the global bioplastic production concerns this segment), while packaging remains the largest field of application for bioplastics since around 54% of the global bioplastic production is used to serve the packaging industry, including shopping bags producers, plastic bottles producers, and food packaging industry.
Biodegradable shopping bags are made of polymers that degrade, or decompose, when exposed to air, water, or sunlight. There are three main types of biodegradable bags, i.e., (1) biodegradable bags made from resins containing starches, polyethylene, and heavy metals such as cadmium, lead, and beryllium, (2) biodegradable bags made by using starches combined with biodegradable polymers such as PLA, and (3) oxo-biodegradable bags, which use Totally Degradable Plastics Additives (TDPAt) to stimulate the breakdown of polymers and thus speed up the biodegradation process of conventional plastics.
As regard to food packaging, in the USA premarketing approval by the Food and Drug Administration is required to ensure that materials are wholesome, safe, and effective [bib_ref] Food packaging regulation in the United States and the European Union, Heckman [/bib_ref]. On the other hand, in Europe, food contact materials regulations sets specific manufacturing goals to assure a good quality control system and specifies the thresholds according to the form and composition of polymers, which shall explicitly be authorized in order to preserve food safety (European Commission, 2006; European Commission 2011). Anyway, biobased materials are mostly used to pack short shelf-life products or long shelf-life ones, which do not need very high oxygen and/or water barrier properties, such as fresh fruits, vegetables, pasta, and chips [bib_ref] Application of bioplastics for food packaging, Peelman [/bib_ref]. Actually, biomaterials available show such a wide range of properties, that they are also applicable as packaging materials for other food products, which request stricter conditions, like Modified Atmosphere Packaging (MAP).
Bioplastic materials also offer several advantages in the agriculture sector. Eight percent of the global production of bioplastics is covered by the agriculture and horticulture segment. Examples of bio-based products used in agriculture are mulching films and pots [bib_ref] Recycled wastes of tomato and hemp fibres for biodegradable pots: Physico-chemical characterization..., Schettini [/bib_ref] [bib_ref] An overview on innovative biodegradable materials for agricultural applications, Malinconico [/bib_ref]. Soil mulching is a practice used in cultivation, which allows weed suppression, reduces the loss of moisture from the soil, and may promote the increasing of soil temperature. Ploughing-in of bio-based and biodegradable mulching films after use instead of collecting them from the field and cleaning off the soil is a more practical and time saving solution. In the same way, bio-based pots are used.
For the automotive field, instead, components made completely or partially from bioplastics can provide a safety standard, that is of ultimate importance in the transportation sector. The products include seat and airbag covers as well as steering wheels. Some of the bio-based plastics such as bio-based polyamides and bio-based polyesters are already successfully being used by leading automotive brands around the world today with the aim of reducing their products' environmental impact. For example, Toyota typically uses bio-based polypropylene/polylactic acid (PP/PLA) composite derived from plant materials for the realization of up to 60% of the interior design of cars.
Biopolymers find applications in several housewares, such as kitchen tools and utensils, washable storage containers and cups, bathroom accessories, toys, hangers, and hooks. For example, hangers from United Colors of Benetton are made of biodegradable polymers. Nontoxic biodegradable polymers are also being used as sutures by surgeons in life-saving heart operations and other procedures. Easily sterilized, the sutures remain strong and intact until the surrounding tissues have healed. The sutures dissolve and are readily metabolized in the body leaving no trace. Moreover, there has been a surge of bioplastic products that are being introduced in the fast-moving consumer electronics sector, such as touch screen computer casings, loudspeakers, keyboard elements, mobile casings, vacuum cleaners, and a mouse for a laptop. SUPLA produced the first bioplastic touch screen computer by using PLA, in collaboration with a Taiwanese company (Kuender).
To date, a lot of companies have been identified as key players in the production of bioplastics and their distribution witnesses that the majority of them are located in Europe. Many of these companies produce sustainable bioplastics made from plant-based renewable resources, like corn, potatoes, and wheat. The land used to grow the renewable feedstock for the production of bioplastics is estimated to be 0.7 million hectares in 2021 and continues to account for 0.015 percent of the global agricultural area of 4.7 billion hectares. Despite the market growth predicted in the next five years, the land use share for bioplastics will only slightly increase to 0.02 percent.
Novamont SpA (www.novamont.com) is one of the major starch bioplastics producers. The trade name of their starch-based bioplastic is "Mater-Bi" and it is provided for a wide range of manufacturers, which use it to make bags, mulching film, disposable tableware, and packaging. Furthermore, Amynova Polymers GmbH (www.amynova.com) is engaged in the production of a starch-based substance named "CropCover". CropCover is an innovative "adhesive" non-toxic, non-combustible, and fully biodegradable applied together with pesticides and foliar fertilizers, in order to reduce their rinsing during heavy rainfall and to guarantee a longer stay time on the plant. Biotec Biologische Naturverpackungen GmbH & Co. KG (www.biotec.de) and Cardia Bioplastics (www.cardiabioplastics.com) produce and sell a new generation of customized thermoplastic materials too, with various functional properties fully biodegradable and compostable according to EN 13432. Moreover, there are companies that exploit waste as feedstock for bioplastics production; an example is NaturePlast (www.natureplast.eu). Since 2015, NaturePlast has been producing and marketing a range of biocomposites consisting of by-products and plant fibers (such as hemp), sourced mostly from the French territory. The objective is to incorporate by-products or local waste materials in different polymers to ensure a circular economy and the reclamation of waste materials.
# Conclusions
The valorization of food waste (FW) can create opportunities to produce new valuable bioplastics, which represent an eco-friendly alternative to conventional petroleum-based plastics. Bioplastics produced from fruits and vegetables waste are compatible with the "circular economy", therefore with "zero waste" or more precisely aiming at a complete use of it; moreover, they could create positive synergies between industry and the agro-food sector, with considerable advantages for environmental pollution. This review highlights that the real challenge is to create new eco-friendly materials from food waste and not from specially grown crops, whose production comes at an environmental cost. As the FW potential as raw material for bioplastics production is well known, such a novel perspective focusing on the overall methods used for the design of biomaterials starting from both fruits and vegetables wastes, provided in this review, should be particularly helpful in the fields of the green chemistry and of the environmental sciences. [fig_ref] Table A1: Cont. [/fig_ref]. Fruits and vegetables wastes used in the last 10 years as raw material for the production of bioplastics. A full description of the methods used for the production of bioplastics (Technology and pre-treatment of the waste, processing), as well as the main physical and mechanical properties of the obtained materials are provided.
## Food waste source technology and pre-treatment processing bioplastic type bioplastic mechanical and physical properties reference
Apple pomace
Apple pomace, either washed with water or not, was powdered. A mixture was prepared containing 2% (w/v) of powder and 7% glycerol (w/w of apple pomace powder) and dissolved in 1% (w/v) of citric acid solution under heating (70 - C) and constant magnetic stirring at 560 rpm. Then, the mixture was poured onto a non-sticky plate for casting. A mixture without glycerol was also used in order to prepare a biofilm. Gustaffson et al. [bib_ref] Development of bio-based films and 3D objects from apple pomace, Gustafsson [/bib_ref] Banana peels were boiled in water for about 30 min; then, they were left to dry and squashed to obtain a paste. 25 g of banana paste were placed in a beaker with 3 mL of HCl (0.1 N) and 2 mL of glycerol and stirred. Subsequently, NaOH (0.1 N) was added in order to neutralize the pH up to 7.
## Casting cellulose based biofilm
Weight loss after 13 days: 0.04 g Difference of weight after swelling test (g): 0.01-0.10
Yaradoddi et al. [bib_ref] Biodegradable plastic production from fruit waste material and its sustainable use for..., Yaradoddi [/bib_ref] Banana peels and pseudostems Banana peels were washed, sliced, and blended. Then, they were grounded and ground sample was heated at 150 - C for 2 h at 30 psi. The obtained paste was hydrolyzed (100 mL/50 g sample) by (HCl, 99%). The sample was filtrated and washed with water. 1000 g of sample were mixed with chlorinated paraffin liquid plasticizers (1:8 of sample), 5% acetic acid (5 mL/100 g sample), 5 mL/100 g of polyvinylchloride, cellulose (25%) and 25% starch powder, 5% toluene Phthalates ester and 10%water. Then, 10 mL/100 g of PVC and glycerine were added. The mixture was heated at 150 - C in the oven for 30 min at 30 psi pressure. Banana peels were immersed in a Na 2 S 2 O 5 solution (1% w/v) for 24 h, oven-dried at 60 - C and milled. 100 g of milled peels were washed three times with ethanol, then washed in 200 mL of acetone. The pectin was extracted with a citric acid solution at pH 2.0 at 87 - C for 160 min and then centrifuged. The supernatant pH was adjusted to 3.5 with KOH, added with ethanol, stirred for 30 min and left to precipitate at 4 - C. The pellet was washed with ethanol 70%, and dried at room temperature. Then, it was stirred and its pH adjusted to 7, and it was again dried and milled. For the extraction of cellulose nanocrystals (CNCs), the alcohol insoluble residue of banana peels was suspended in a mixture of 93 wt% acetic acid and 0.3 wt% HCl in distilled water. Subsequently, the pulp was rinsed and washed in more steps and an acid hydrolysis was conducted with a 30 v% H 2 SO 4 solution at 45 - C, for 150 min. After centrifugation-dilution-sonication cycles, the CNC suspension was dialyzed against deionized water. CNCs (at different wt%), 4.5 g of pectin, 1.35 g glycerol, citric acid, and distilled water were mixed to form biofilms. Oliveira et al. [bib_ref] Bionanocomposite films based on polysaccharides from banana peels, Oliveira [/bib_ref] 300 g of banana peels were dipped in acetic acid solution and then placed into a beaker containing 800 mL water and boiled for 30 min. The water was decanted off and the peels were left to dry. The banana peels were pureed. To 25 mL of the paste, 3 mL of 0.5 M HCl and 2 mL of 15% glycerol solution were added. The mixture was stirred, 3 mL of 1% corn starch and 3 mL of 0.5 M NaOH were added to the mixture and stirred again. Banana pseudostem was sliced, dryer at 50 - C for 5 h and milled. Then, 10 g of pseudo-stem flour were soaked in 300 mL of 5% KOH, centrifuged and bleached with 200 mL of 1% NaClO 2 at pH 5 for 1 h in 70 - C. 9 g of the bleached pseudo-stem were mixed with TEMPO solution and 22.5 mL of 12% NaClO. At the end of the reaction, the mixture was homogenized and sonicated. Water containing 0.7% solid nanocellulose was mixed with glycerol, nano-clay or graphene oxide in different proportions. Faradilla et al. [bib_ref] Effect of glycerol, nanoclay and graphene oxide on physicochemical properties of biodegradable..., Faradilla [/bib_ref] Banana peels were boiled for 60 min and then left to dry and blended. In order to obtain a chemical-based material, 100 g of banana paste were mixed with 12 mL of HCl, 8 mL of glycerol and 12 mL of NaOH. The mixture was stirred for 5 min. Alternatively, a natural-based material was obtained by mixing 40 g of banana peels paste with, 1 g of sage, 12 g of glycerol, 12 g of potato starch, 12 g of corn starch and 38 g of water. The mixture was dried using the oven at a temperature of 120 - C for 3-4 h. Azieyanti et al. [bib_ref] Mechanical and Morphology Studies of Bioplastic-Based Banana Peels, Azieyanti [/bib_ref] Banana peels were boiled in water for about 30 min. Water was decanted and the peels were left to dry and then they were squashed to obtain a uniform paste. 25 g of banana paste were mixed with 3 mL of (0.1 N) HCl, 2 mL of glycerol and then 3 mL of 0.1 N NaOH to neutralize the pH up to 7.
Casting Starch based biofilm Not reported Rizwana Beevi et al. [bib_ref] Bioplastic Synthesis Using Banana Peels And Potato Starch And Characterization, Beevi [/bib_ref] Perotto et al. [bib_ref] Bioplastics from vegetable waste: Via an eco-friendly water-based process, Perotto [/bib_ref] 100 g of cassava peels were washed and soaked in sodium metabisulphyte 1%. Then, they were crushed with 100 mL of water. Slurry resulted was extracted with water (ratio of 1:1) two times. The extracts were precipitated for 3 h. The precipitate was dried; then 5 g were mixed with glacial acetic acid 1%, chitosan (20-50%), glycerol (30%), liquid smoke (0-2 mL) and stirred at 70 - C. Fathanah et al. [bib_ref] Characterization of bioplastic based from cassava crisp home industrial waste incorporated with..., Fathanah [/bib_ref] Cassava peels 5.0 g of dried cassava peels waste were mixed with 1.5 mL of glycerol, 0.5 mL of kaffir lime essential oil and 0.7 g of citric acid. The mixture was stirred for 45 min, and heated to 80 - C. Two other samples were prepared without essential oil and without citric acid, respectively.
## Casting
Starch based biofilm Tensile strength (N/cm): 0.3-2. [bib_ref] CO 2 and N 2 O from water resource recovery facilities: Evaluation..., Caniani [/bib_ref] Masruri et al. [bib_ref] The effect of kaffir lime (Citrus hystrix DC) essential oil on bioplastic..., Masruri [/bib_ref] Cassava peels were mashed into a pulp. Then, 10 g were extracted with 50 mL of water. The extract was washed with water. Then, the juice precipitated and dried under direct sunlight to form a flour or starch. 3 g of starch were mixed with glycerol (25% wt)) and chitosan (2 and 3% wt). The mixture was heated at a 80-90 - C and stirred. Citrus peel derived pectin powder was mixed with glycerol (30% (w/w)) for 2 min to get a uniform dough which was then formed into ball shape (total weight of 2.5 g). The obtained blend was placed in molding press. The compression molding process was performed for 10 min under operation conditions of 1.33 MPa and 120 - C.
Pectin-based biomass films were also produced by incorporation of lyophilized and milled fungal biomass. Biomass concentrations varied in the range of 0-35% of the total mixture. Glycerol content was kept at 30%. Jackfruit seeds Jackfruit seeds were removed from the skin of the arrows and then washed and powdered. The obtained powder was mixed with sorbitol (from 0 to 6 mL) and poly vinyl alcohol (from 0 to 3 g).
## Molding
Not specified Not specified Tensile strength (MPa): 0-~2.2 Elongation at break: 0-7% Lestari et al. [bib_ref] Bioplastic from Jackfruit Seeds and Rice, Lestari [/bib_ref] Moro et al. [bib_ref] Bioplastics of Native Starches Reinforced with Passion Fruit Peel, Moro [/bib_ref] Peanut hulls Peanut hulls, were stored at 4 - C, and then reduce to powder. The mixture including 17 g of peanut hulls mashed to a size of around 100 microns was blended minutes, gradually adding potato flour from skins (30 g), whole milk (48 mL) and glycerol (5 mL). The mixed ingredients formed a compound that was cooked in a fan-assisted oven at 180 - C for 13 min.
Casting Biocomposite Weight loss %: 6.5 ± 0.5 Troiano et al. [bib_ref] DIY Bioplastics from Peanut Hulls Waste in a Starch-Milk Based Matrix, Troiano [/bib_ref] Pineapple leaf Pineapple leaf were scaffed and then the fibers were cut into small sizes and grinded. The grinded fibers were then sieved to get the highest amount of fiber length available. Then they were placed in oven for 24 h and subsequently mixed with tapioca-based bioplastic resin (70-90% w/w).
The mixture was first extruded into pellet at 160 - C. The pellets were then placed in the mold and hot pressed at 160 - C for 5 min at 8 MPa, and then cold pressed at room temperature for 5 min at 8 MPa. Bilo et al. [bib_ref] A sustainable bioplastic obtained from rice straw, Bilo [/bib_ref] Rice straw was dried, powdered and cooked for 8 h at 80 - C. 10% NaOH solution was added and the mixture was autoclaved for 15 min at 121 - C. The obtained mixture was added with 10% sodium hypochlorite processed using ultrafine grinder in wet milling method. 2% cellulose was mixed with 2 L of water and passed through the grinder for up to 30 cycles until a nanocellulose gel was formed. 5% of the produced nanocellulose was introduced in the Enviplast ® formula. Iriani et al. [bib_ref] The effect of agricultural waste nanocellulose on the properties of bioplastic for..., Iriani [/bib_ref] Rice straw were chopped to prepare the fibres with lengths ranging from 0.5 to 2 mm. The fibers were dehydrated for 24 h within a vacuum oven at 105 - C. Three chosen polymers (PLA, Lignin, PP) were separately compounded with 20% of rice straw fiber.
## Extrusion
Biocomposite Not reported Dahy et al.Rice waste powdered. The powder of rice waste was mixed with chitosan (from 30 to 60%) and glycerol (from 0 to 3 mL), heated at 50-60 - C for 30 min.
## Molding cellulose based biofilm
Tensile strength (MPa):~0-60 Elongation at break: 2-4% Water resistance: 0.1-0.6%
Lestari et al. [bib_ref] Bioplastic from Jackfruit Seeds and Rice, Lestari [/bib_ref] Yacob et al. [bib_ref] Effect of cellulose fiber loading on the properties of starch-based films, Yacob [/bib_ref] Soy waste Soy waste was bleached with a solution of distilled water:sodium hypochlorite (70:30). Then, it was separated from the solvent, rinsed with distilled water and dried in the oven for one hour at 100 - C. Subsequently, it was powdered and 3.0 g were mixed with corn starch (9.5 g), glycerol (5 mL), vinegar (5 mL) and water (60 mL). The mixing process was carried out at 25 - C and 50 rpm for 10 min.
## Casting biocomposite
Maximum value of force before fracture (N): 6.71 Water absorption (%): 114.17
Muhammad et al. [bib_ref] Development of bio based plastic materials for packaging from soybeans waste, Muhammad [/bib_ref] Spinach steams Spinach steams were washed to remove residual sugars and alcohols and then dried in an oven at 40 - C overnight. Solutions of 3% by weight of solids in TFA were prepared in glass vials. Vials were sealed with Parafilm and were placed in a benchtop lab shaker for 29 days. The obtained solution was centrifuged in order to remove any residuals.
## Casting cellulose based biofilm
Tensile stress at break (MPa): 1 Water adsorption %: <10 (at a relative humidity <50%) Water adsorption %: >10 (at a relative humidity >80%) Initial decomposition temperature under 44% of relative humidity ( - C):~130
Bayer et al. [bib_ref] Direct transformation of edible vegetable waste into bioplastics, Bayer [/bib_ref] Tea leaves waste Tea leaves waste were dried under vacuum at 70 - C, grounded, and sifted. Tea waste powder (TW) was dried under vacuum at 70 - C. TW bioplastics were synthesized with 1 g of TW powder in 20 mL of 3% citric acid solution (TW-CA) or only with water (TW-H2O). Carboxymethylcellulose sodium salt at 5% was investigated also as an additive to TW bioplastics (TW-CMC). All samples were magnetically stirred in an oil bath at 60 - C for 12 h, then casted. Heredia-Guarreiro et al. [bib_ref] Sustainable polycondensation of multifunctional fatty acids from tomato pomace agro-waste catalyzed by..., Heredia-Guerrero [/bib_ref]
[fig] Figure 1: Scheme of bioplastics classification: [/fig]
[fig] Figure 2: The main three steps through which the biodegradation of polymers occurs: (1) bio-deterioration, (2) bio-fragmentation, and (3) assimilation. [/fig]
[fig] Figure 3: Effects of the plasticizers' addition on mechanical and physical properties of bioplastic materials. [/fig]
[fig] Figure 4: Figure 4. Conversion of food wastes into bioplastics could occur through biopolymers extraction and their mechanical manufacturing (extrusion, casting, molding, or combination of them). In more complex processes, food wastes are used as substrate for bacterial fermentation in order to produce biopolymers or as raw material for the extraction of other natural components, such as fibers, which act as reinforcing agents and/or natural filler of biocomposites. [/fig]
[fig] Author: Contributions: Conceptualization, M.A.A.; investigation, M.A.A.; writing-original draft preparation, M.A.A.; review and editing, R.P.; supervision, G.M., M.B., and G.B. All authors have read and agreed to the published version of the manuscript.Funding: This research received no external funding. Informed Consent Statement: Not applicable. Data Availability Statement: Data sharing not applicable.Conflicts of Interest:The authors declare no conflict of interest. [/fig]
[table] Table A1: Cont. [/table]
[bib_ref] From waste plastics to industrial raw materials: A life cycle assessment of..., Gu [/bib_ref] |
Settlers of our inner surface – factors shaping the gut microbiota from birth to toddlerhood
One sentence summary: This review takes an ecological perspective to guide readers through the most important factors controlling the succession and establishment of gut microbiota in infants after birth. Editor: Jan Roelof van der Meer † Martin Frederik Laursen, http://orcid.org/0000-0001-6017-7121 ‡ Martin Iain Bahl, http://orcid.org/0000-0003-1579-8038 § Tine Rask Licht, http://orcid.org/0000-0002ABSTRACTDuring the first 3 years of life, the microbial ecosystem within the human gut undergoes a process that is unlike what happens in this ecosystem at any other time of our life. This period in time is considered a highly important developmental window, where the gut microbiota is much less resilient and much more responsive to external and environmental factors than seen in the adult gut. While advanced bioinformatics and clinical correlation studies have received extensive focus within studies of the human microbiome, basic microbial growth physiology has attracted much less attention, although it plays a pivotal role to understand the developing gut microbiota during early life. In this review, we will thus take a microbial ecology perspective on the analysis of factors that influence the temporal development of the infant gut microbiota. Such factors include sources of microbes that seed the intestinal environment, physico-chemical (abiotic) conditions influencing microbial growth and the availability of nutrients needed by the intestinal microbes.
# Introduction
The gut microbiota in adults comprises a large variety of organisms including bacteria, fungi, archaea, protists and viruses. The bacteria vastly outnumber the other groups, and will have our focus in the present review. As most observations about intestinal microbes are based on fecal samples, we use the words 'gut' or 'intestinal' to refer to observations based on feces.
The development of the gut microbiota in an infant undergoes drastic changes: Shortly after birth, only a limited number of living microbes represented by a few species are present in the gut, while a load of up to 10 12 bacteria per gram and about 150-200 bacterial species can be found later in infancy [bib_ref] The first microbial colonizers of the human gut: composition, activities, and health..., Milani [/bib_ref] [bib_ref] Temporal development of the gut microbiome in early childhood from the TEDDY..., Stewart [/bib_ref].
Reported correlations of the characteristics of the early life microbiota with specific health or disease features in childhood or adulthood include a multitude of conditions, of which many are related to atopy [bib_ref] Neonatal gut microbiota associates with childhood multisensitized atopy and T cell differentiation, Fujimura [/bib_ref] [bib_ref] Window-of-opportunity: neonatal gut microbiota and atopy, Johnson [/bib_ref]. However, for most conditions it remains a challenge to establish causal evidence of a microbial influence on later health [bib_ref] The first microbial colonizers of the human gut: composition, activities, and health..., Milani [/bib_ref]. Here, we will focus on the external and environmental factors of the gut that affect microbial exposure and growth physiology and thereby shape the microbial ecosystem and the temporal succession of bacterial establishment, which we view as a fundament for understanding the role of gut bacteria in host health.
In order for a given organism to establish in a given ecosystem, first, it needs to get there, second, it needs to find an ecological niche in terms of conditions and nutrients, which allows it to proliferate and third, it needs to be able to be competitive within this niche. Unfolding this perspective, we will discuss the sources of microbial seeding to the infant gut, the intestinal physicochemical (abiotic) conditions governing microbial growth and the role of nutrient availability and bacterial cross-feeding and competition for nutritional and topographical niches . It is important to note that many of these abiotic factors may in themselves be confounders influencing (or reflecting) human health through mechanisms that are independent of the gut microbes. However, this does not make it less important to be aware of them, since they may often be key to understanding observed associations between infant gut microbiota and risk of disease.
## Sources of microbial seeding
The early-life gut microbiota consists of microbes that are acquired either vertically or horizontally. Studies with captured wild mice of different origins, inbred and co-housed (same room, not co-caged) for up to 11 generations, suggest that transmission of gut bacteria occurs primarily vertically (across generations within mouse origin) and to lesser extent horizontally (across mouse origin along multiple generations; [bib_ref] Transmission modes of the mammalian gut microbiota, Moeller [/bib_ref]. The microbes reported to be transmitted vertically were typically strictly anaerobic clostridia and bacteroidia, whereas horizontally transmitted bacteria were usually facultative anaerobic bacilli [bib_ref] Transmission modes of the mammalian gut microbiota, Moeller [/bib_ref]. Thus, taxonomic and phenotypic characteristics of bacteria may affect their transmission mode and it seems that maternal/parental microbiomes are the most pronounced sources of bacteria transmitted to vaginally born offspring. Indeed, in humans, maternal body sites has been reported to contribute with approximately 50% of the bacterial species found in the infant gut throughout the first 4 months of life [bib_ref] Mother-to-infant microbial transmission from different body sites shapes the developing infant gut..., Ferretti [/bib_ref]. Still, horizontally acquired bacteria e.g. from siblings [bib_ref] Having older siblings is associated with gut microbiota development during early childhood, Laursen [/bib_ref] or unrelated individuals , household pets [bib_ref] Cohabiting family members share microbiota with one another and with their dogs, Song [/bib_ref] [bib_ref] Furry pets modulate gut microbiota composition in infants at risk for allergic..., Nermes [/bib_ref] [bib_ref] Joint effects of pregnancy, sociocultural, and environmental factors on early life gut..., Levin [/bib_ref] , the infant's (complementary) diet [bib_ref] Infant gut microbiota development is Driven by transition to family foods independent..., Laursen [/bib_ref] and the environment [bib_ref] The first microbial environment of infants born by C-section: the operating room..., Shin [/bib_ref] [bib_ref] Strain-resolved analysis of hospital rooms and infants reveals overlap between the human..., Brooks [/bib_ref] contribute to the establishment of the complex infant gut microbiota. Further, transmission of bacteria from the infant's own oral microbiota, especially in the neonatal period, has been found to be a significant source of bacteria to the infant gut [bib_ref] Mother-to-infant microbial transmission from different body sites shapes the developing infant gut..., Ferretti [/bib_ref] [bib_ref] Extensive transmission of microbes along the gastrointestinal tract, Schmidt [/bib_ref].
## Vertically transmitted bacteria
A number of recent papers have strongly suggested that extensive vertical transmission of microbes to the infant gut occurs [bib_ref] An integrated metagenomics pipeline for strain profiling reveals novel patterns of bacterial..., Moossavi [/bib_ref] [bib_ref] Maturation of the infant microbiome community structure and function across multiple body..., Chu [/bib_ref] [bib_ref] Mother-to-infant microbial transmission from different body sites shapes the developing infant gut..., Ferretti [/bib_ref] [bib_ref] Strain-level analysis of mother-to-child bacterial transmission during the first few months of..., Yassour [/bib_ref] [bib_ref] Discordant transmission of bacteria and viruses from mothers to babies at birth, Maqsood [/bib_ref]. The sources of microbes for the infant gut include maternal vaginal, oral, gut, skin and breast milk microbial communities and data indicate that these together contribute with the majority of species that are establishing in the infant gut [bib_ref] Maturation of the infant microbiome community structure and function across multiple body..., Chu [/bib_ref] [bib_ref] Association between breast milk bacterial communities and establishment and development of the..., Pannaraj [/bib_ref] [bib_ref] Mother-to-infant microbial transmission from different body sites shapes the developing infant gut..., Ferretti [/bib_ref] [bib_ref] Discordant transmission of bacteria and viruses from mothers to babies at birth, Maqsood [/bib_ref]. Especially the maternal gut [bib_ref] Mother-to-infant microbial transmission from different body sites shapes the developing infant gut..., Ferretti [/bib_ref] [bib_ref] Discordant transmission of bacteria and viruses from mothers to babies at birth, Maqsood [/bib_ref] and breast milk [bib_ref] Association between breast milk bacterial communities and establishment and development of the..., Pannaraj [/bib_ref] are influential sources, whereas seeding from maternal oral, vaginal and skin microbiota seem to be mainly significant in the neonatal phase [bib_ref] Maturation of the infant microbiome community structure and function across multiple body..., Chu [/bib_ref] [bib_ref] Mother-to-infant microbial transmission from different body sites shapes the developing infant gut..., Ferretti [/bib_ref]. In that regard, the heterogeneity often observed in the neonatal gut microbial communities [bib_ref] Dynamics and stabilization of the human gut microbiome during the first year..., Bäckhed [/bib_ref] [bib_ref] Maturation of the infant microbiome community structure and function across multiple body..., Chu [/bib_ref] seem to be linked to an initial individual pattern of similarity to the vaginal, oral or skin microbiota of the mother, probably ascribed to transient colonization by microbes transmitted from these sources [bib_ref] Mother-to-infant microbial transmission from different body sites shapes the developing infant gut..., Ferretti [/bib_ref].
## Vaginal microbes
The vaginal microbiota is remarkably stable over the course of pregnancy [bib_ref] Temporal and spatial variation of the human microbiota during pregnancy, Digiulio [/bib_ref] , and has been grouped into five distinct community types, either dominated by Lactobacillus crispatus (type I), Lactobacillus gasseri (type II), Lactobacillus iners (III) or Lactobacillus jensenii (type V) or a type IV characterized by high diversity and a mixture of Prevotella, Sneathia, Gardenella, Atopobium and Megasphaera spp. [bib_ref] Vaginal microbiome of reproductiveage women, Ravel [/bib_ref] [bib_ref] Temporal dynamics of the human vaginal microbiota, Gajer [/bib_ref] [bib_ref] Temporal and spatial variation of the human microbiota during pregnancy, Digiulio [/bib_ref]. Interestingly, the relative abundance of bacterial species in the vagina prior to birth does not seem to be coupled to the chance of vertical transmission [bib_ref] Ecological succession in the vaginal microbiota during pregnancy and birth, Rasmussen [/bib_ref]. Nevertheless, vaginally delivered newborns initially harbor a gut microbiota dominated by Lactobacilli, Prevotella and Sneathia spp., resembling the corresponding mothers vaginal microbiota [bib_ref] Delivery mode shapes the acquisition and structure of the initial microbiota across..., Dominguez-Bello [/bib_ref] , while, C-section delivered newborns are initially colonized by typical (maternal) skin microbes such as Propionibacterium, Staphylococcus and Corynebacterium [bib_ref] Delivery mode shapes the acquisition and structure of the initial microbiota across..., Dominguez-Bello [/bib_ref]. All of the four major vaginal lactobacilli; L. iners, L. crispatus, L. gasseri and L. jensenii but also Gardenella vaginalis and Atopobium vaginae, have been found to be transmitted but colonize only transiently in the infant [bib_ref] Mother-to-infant microbial transmission from different body sites shapes the developing infant gut..., Ferretti [/bib_ref]. Within the first day of life, about 16.3% of the neonatal gut microbiota is composed of maternal vaginal species, but this rapidly declines within the first week [bib_ref] Mother-to-infant microbial transmission from different body sites shapes the developing infant gut..., Ferretti [/bib_ref] , indicating that the vaginal microbes persist only transiently in the infant gut environment. Interestingly, multiple studies have shown a depletion of Bifidobacterium and Bacteroides species in the gut of C-section compared to vaginally born infants, spanning the first year of life [bib_ref] Dynamics and stabilization of the human gut microbiome during the first year..., Bäckhed [/bib_ref] [bib_ref] Temporal development of the gut microbiome in early childhood from the TEDDY..., Stewart [/bib_ref]. However, these are not common vaginal microbes , but can be restored by oral transplantation with bacteria from the maternal gut [bib_ref] Maternal fecal microbiota transplantation in cesarean-born infants rapidly restores normal gut microbial..., Korpela [/bib_ref] , which unlike the vaginal microbes are adapted for the intestinal environment. It is therefore likely that the lack of exposure to the maternal rectal environment is the main cause of the observed lower levels of these taxa in infants born by C-section.
## Skin and oral microbes from the mother
The human skin is predominantly colonized by Staphylococcus, Corynebacterium and Propionibacterium [bib_ref] The human skin microbiome, Byrd [/bib_ref] , and the areolar/nipple skin is often additionally colonized by Streptococcus [bib_ref] The influence of different maternal microbial communities on the development of infant..., Drell [/bib_ref] [bib_ref] Association between breast milk bacterial communities and establishment and development of the..., Pannaraj [/bib_ref] ; note that Propionibacterium is now designated Cutibacterium [bib_ref] The natural history of cutaneous propionibacteria, and reclassification of selected species within..., Scholz [/bib_ref]. However, for clarity, we will comply with the genus names used in the cited literature). Species found to be shared between maternal skin and infant gut in the neonatal period include Streptococcus spp., Staphylococcus epidermidis and Propionibacterium acne [bib_ref] Mother-to-infant microbial transmission from different body sites shapes the developing infant gut..., Ferretti [/bib_ref]. While the breast skin microbiota contributes with 5-6.8% of the bacterial species found in the infant gut during the first days of life, this declines to 3.2% after 1 week, and after 4 months bacteria originating from Figure 1. Influence of selected factors on the developing microbiota. In the first period after birth, the bacterial load in the intestine is low, meaning that many ecological niches are free for the seeded bacteria to explore. The high inter-individual (beta) diversity at this stage is probably reflecting a high influence from the many different sources of seeding to the gut at this stage. When the complexity of diet increases, so does the complexity (alpha diversity) of the bacterial community, and eventually the impact of original seeding sources is no longer detectable. The increasing hostility of the gut environment, characterized by reduced oxygen availability and reduced pH and later by scarceness bacterial of nutrients subject to competition, selects for a community that is optimized for coping with this, thereby reducing the beta diversity, and increasing the impact of competition. (The figure was created in BioRender). maternal skin are no longer detectable in the infant gut [bib_ref] Mother-to-infant microbial transmission from different body sites shapes the developing infant gut..., Ferretti [/bib_ref].
Abundant members of the oral microbiota include species within Streptococcus, [bib_ref] Strong multivariate relations exist among milk, oral, and fecal microbiomes in mother-infant..., Williams [/bib_ref]. Maternal tongue dorsum bacterial species are also found in infant gut throughout the first 4 months of life, accounting for up to 16% of the infant gut microbiota [bib_ref] Mother-to-infant microbial transmission from different body sites shapes the developing infant gut..., Ferretti [/bib_ref]. Here, Streptococcus parasanguinis, Rothia mucilaginosa, Prevotella melaninogenica, Haemophilus parainfluenzae and Veillonella parvula are some of the most commonly species found in both ecosystems [bib_ref] Mother-to-infant microbial transmission from different body sites shapes the developing infant gut..., Ferretti [/bib_ref]. However, it remains a challenge to distinguish strains originating from the mother's oral environment from the infants own oral bacteria. Thus, the apparent sharing of species between maternal oral and infant gut ecosystems may result from seeding of infant's own oral strains into the gut [bib_ref] Mother-to-infant microbial transmission from different body sites shapes the developing infant gut..., Ferretti [/bib_ref].
## Fecal microbes from the mother
The adult gut microbiota is dominated by Clostridiales and Bacteroidales, and lower proportions of genera within the Enterobacteriales and Bifidobacteriales [bib_ref] Enterotypes of the human gut microbiome, Arumugam [/bib_ref]. During pregnancy the microbiota undergoes significant changes including a decrease in alpha diversity and relative increases in Enterobacteriaceae, Streptococcus and Bifidobacterium species [bib_ref] Host remodeling of the gut microbiome and metabolic changes during pregnancy, Koren [/bib_ref]. Interestingly, only a limited group of maternal genera appear to be transmitted to the infant, mostly confined to Bifidobacterium, Bacteroides, Escherichia and Streptococcus spp. [bib_ref] Dynamics and stabilization of the human gut microbiome during the first year..., Bäckhed [/bib_ref] [bib_ref] Mother-to-infant microbial transmission from different body sites shapes the developing infant gut..., Ferretti [/bib_ref] [bib_ref] Discordant transmission of bacteria and viruses from mothers to babies at birth, Maqsood [/bib_ref]. Nevertheless, at the species level, 20-50% of the bacteria present in the infant gut during the first 4 months of life are shared with the maternal gut microbiota [bib_ref] Mother-to-infant microbial transmission from different body sites shapes the developing infant gut..., Ferretti [/bib_ref]. While sharing of bacteria at species level within mother-infant pairs increase with infant age, sharing at strain level tends to decrease [bib_ref] An integrated metagenomics pipeline for strain profiling reveals novel patterns of bacterial..., Moossavi [/bib_ref] , suggesting that strains acquired from the mother gradually get replaced by horizontally acquired strains of the same species. However, within some species, vertically transmitted strains are more likely to persist and stably colonize the infant gut, than horizontally acquired strains [bib_ref] An integrated metagenomics pipeline for strain profiling reveals novel patterns of bacterial..., Moossavi [/bib_ref] [bib_ref] Mother-to-infant microbial transmission from different body sites shapes the developing infant gut..., Ferretti [/bib_ref]. These include strains of Bacteroides and Escherichia (Escherichia coli). Noteworthy, these strains are typically highly abundant colonizers of the infant gut [bib_ref] Transmission of intestinal Bifidobacterium longum subsp. longum strains from mother to infant,..., Makino [/bib_ref] [bib_ref] Exploring vertical transmission of bifidobacteria from mother to child, Milani [/bib_ref] [bib_ref] An integrated metagenomics pipeline for strain profiling reveals novel patterns of bacterial..., Moossavi [/bib_ref] [bib_ref] Mother-to-infant microbial transmission from different body sites shapes the developing infant gut..., Ferretti [/bib_ref] [bib_ref] Strain-level analysis of mother-to-child bacterial transmission during the first few months of..., Yassour [/bib_ref]. Thus, although only a minor selection of the maternal microbes are transmitted, they constitute a significant amount of the total bacterial population in the infant gut.
## Microbes from breast milk
Human breast milk contains on average 10 3 (range 10 1 -10 5 ) bacteria per mL [bib_ref] Impact of human milk bacteria and oligosaccharides on neonatal gut microbiota establishment..., Jost [/bib_ref]. As breastfed infants consume 600-1200 mL milk per day [bib_ref] Excessive weight gain followed by catch-down in exclusively breastfed infants: an exploratory..., Larsson [/bib_ref] , this results in a significant exposure of up to 10 7 bacteria daily. Microbes found in breast milk primarily originate from maternal skin, infant oral cavity and from the environment (Moossavi and Azad 2019; [bib_ref] Strong multivariate relations exist among milk, oral, and fecal microbiomes in mother-infant..., Williams [/bib_ref] [bib_ref] Contributions to human breast milk microbiome and enteromammary transfer of Bifidobacterium breve, Kordy [/bib_ref]. Although still subject to debate, internal routes of transfer including enteromammary [bib_ref] The origin of human milk bacteria: is there a bacterial entero-mammary pathway..., Rodriguez [/bib_ref] and oro-mammary (Moossavi and Azad 2019) pathways have been speculated to affect the occurrence of microbes in breastmilk. Prevalent and abundant bacteria found in breast milk include skin-associated bacteria such as Staphylococcus, Propionibacterium and Corynebacterium spp. [bib_ref] Systematic review of the human milk microbiota, Fitzstevens [/bib_ref] [bib_ref] Evaluation of human milk microbiota by 16S rRNA gene Next-Generation Sequencing (NGS)..., Treven [/bib_ref] , oral cavityassociated Streptococcus, Veillonella, Rothia and Gemella [bib_ref] Impact of human milk bacteria and oligosaccharides on neonatal gut microbiota establishment..., Jost [/bib_ref] [bib_ref] Evaluation of human milk microbiota by 16S rRNA gene Next-Generation Sequencing (NGS)..., Treven [/bib_ref] [bib_ref] Strong multivariate relations exist among milk, oral, and fecal microbiomes in mother-infant..., Williams [/bib_ref] , but also bacteria likely to be of environmental origin, such as Acinetobacter and Pseudomonas spp. [bib_ref] Microbial communities in human milk relate to measures of maternal weight, Lundgren [/bib_ref]. Furthermore gut-associated species such as Lactobacillus, Enterococcus and Bifidobacterium spp. are commonly isolated from breast milk, although in low abundance [bib_ref] Impact of human milk bacteria and oligosaccharides on neonatal gut microbiota establishment..., Jost [/bib_ref]. During the first month of life, 28% of the infant gut microbial population, at species level, is shared with the maternal breast milk microbiota [bib_ref] Association between breast milk bacterial communities and establishment and development of the..., Pannaraj [/bib_ref]. Species found to be shared within mother-infant dyads include Staphylococcus, Streptococcus, Veillonella, Rothia, Enterococcus, Lactobacillus and Bifidobacterium spp. [bib_ref] Transmission of intestinal Bifidobacterium longum subsp. longum strains from mother to infant,..., Makino [/bib_ref] [bib_ref] Association between breast milk bacterial communities and establishment and development of the..., Pannaraj [/bib_ref] , and isolation of identical strains within these genera has been demonstrated [bib_ref] Human milk is a source of lactic acid bacteria for the infant..., Martin [/bib_ref] [bib_ref] Sharing of bacterial strains between breast milk and infant feces, Martín [/bib_ref]. Transmission of Bifidobacterium spp. via breast milk has additionally convincingly been shown by combining gene marker based amplicon sequencing, metagenomic analysis and strain isolation in mother infant-pairs [bib_ref] Transmission of intestinal Bifidobacterium longum subsp. longum strains from mother to infant,..., Makino [/bib_ref] [bib_ref] Exploring vertical transmission of bifidobacteria from mother to child, Milani [/bib_ref]. Importantly, B. longum and B. breve strains detected in both maternal breast milk and initial infant feces persistently colonize the infant gut for up to 6 months [bib_ref] Transmission of intestinal Bifidobacterium longum subsp. longum strains from mother to infant,..., Makino [/bib_ref] [bib_ref] Exploring vertical transmission of bifidobacteria from mother to child, Milani [/bib_ref]. Transfer of bacteria via breast milk, especially lactic acid bacteria and bifidobacteria, thus constitutes an important mechanism by which infants acquire bacteria that colonize their gastrointestinal tract.
## Horizontally transmitted bacteria
In addition to microbes acquired from the maternal sources described above, the infant is exposed to a variety of other microbes from the environment including siblings, pets, surfaces in the home and in the hospital and from dietary sources. Exposure to new microbes thus increases with increasing age of the infant, however the influence of such exposure on the composition of the gut microbiota decreases as the child grows older and available ecological niches in the gut get occupied .
## Microbes from siblings, pets and other environmental sources
The presence of siblings clearly impacts the composition of the infant gut microbiota [bib_ref] Having older siblings is associated with gut microbiota development during early childhood, Laursen [/bib_ref] [bib_ref] Early-life events, including mode of delivery and type of feeding, Siblings and..., Martin [/bib_ref] [bib_ref] Temporal development of the gut microbiome in early childhood from the TEDDY..., Stewart [/bib_ref] , and the strongest influence is observed during mid-late infancy and early toddlerhood, i.e. between 6 and 18 months of age [bib_ref] Temporal development of the gut microbiome in early childhood from the TEDDY..., Stewart [/bib_ref]. Microbial richness in toddlers increases with increasing numbers of older siblings [bib_ref] Having older siblings is associated with gut microbiota development during early childhood, Laursen [/bib_ref].
An important species, Faecalibacterium prausnitzii, which is considered a marker of gut health [bib_ref] Ecology and metabolism of the beneficial intestinal commensal bacterium Faecalibacterium prausnitzii, Miquel [/bib_ref] , is very prevalent and abundant in the adult gut but virtually nondetectable in breastfed infants before age 4-6 months. The increase in abundance and prevalence of this species seems to be accelerated by the presence of older siblings . Also, the Bifidobacterium catenulatum group shows a faster increase in prevalence during the first 6 months of life in infants with older siblings as compared to first-borns [bib_ref] Early-life events, including mode of delivery and type of feeding, Siblings and..., Martin [/bib_ref].
Several reports demonstrate that also pets in the household represent a source of microbial seeding to the infant gut [bib_ref] Cohabiting family members share microbiota with one another and with their dogs, Song [/bib_ref] [bib_ref] Furry pets modulate gut microbiota composition in infants at risk for allergic..., Nermes [/bib_ref]. This builds on correlations between household pets and microbial diversity ), but also on evidence of increases in specific bacterial taxa in children exposed to pets [bib_ref] Furry pets modulate gut microbiota composition in infants at risk for allergic..., Nermes [/bib_ref] , or of the pet serving as a 'vehicle' for transmission of microbes between humans in the household [bib_ref] Cohabiting family members share microbiota with one another and with their dogs, Song [/bib_ref]. An example is the animal-derived Bifidobacterium pseudolongum, which is more prevalent in infants who have been exposed to pets, than in controls [bib_ref] Furry pets modulate gut microbiota composition in infants at risk for allergic..., Nermes [/bib_ref].
Finally, although many measures are taken to protect newborns from nosocomial infections, the hospital environment encountered by the infant immediately after birth have also been suggested to represent a source of microbial seeding [bib_ref] The first microbial environment of infants born by C-section: the operating room..., Shin [/bib_ref]. However, solid proof of transmission of specific strains to the infant gut requires advanced sequence-based methods in order to exclude that given measures of overlap are indeed reflecting occurrence of the same clones, and do not merely reflect parallel occurrence of different clones of the same species in the gut and surroundings, respectively. Indeed, identical strains assembled from metagenomes of hospitalized preterm infant feces and the corresponding hospital room environment has been demonstrated [bib_ref] Strain-resolved analysis of hospital rooms and infants reveals overlap between the human..., Brooks [/bib_ref]. These include strains of opportunistic pathogenic species such as Klebsiella pneumoniae, Staphylococcus epidermidis, Enterococcus faecalis and Pseudomonas aeruginosa. While these are important colonizers of the hospitalized preterm infant [bib_ref] Persistent metagenomic signatures of early-life hospitalization and antibiotic treatment in the infant..., Gasparrini [/bib_ref] , they are probably less important sources for the term born neonate with only short hospitalization. In this context, it should be noted that the gut microbiota of preterm infants differs from that of healthy infants in many other ways, which have been the topic of a number of important recent studies [bib_ref] Persistent metagenomic signatures of early-life hospitalization and antibiotic treatment in the infant..., Gasparrini [/bib_ref] [bib_ref] The preterm gut microbiota: an inconspicuous challenge in nutritional neonatal care, Henderickx [/bib_ref] [bib_ref] Gut microbiota development of preterm infants hospitalised in intensive care units, Tauchi [/bib_ref] , but is not within our scope here.
## Microbes ingested with diet
A number of formula and dietary products for infants are fortified with lactic acid bacteria (probiotics) intended to beneficially influence gut health. However, typically these strains do not establish in the infant gut and can only be detected in feces during and shortly after the period of repeated ingestion [bib_ref] Effect of a milk formula containing probiotics on the fecal microbiota of..., Mah [/bib_ref] [bib_ref] Fate, activity, and impact of ingested bacteria within the human gut microbiota, Derrien [/bib_ref]. Once the child becomes habituated to foods that have not been boiled or otherwise sterilized, the microbes residing on such foods will evidently end up in the intestinal tract. It is beyond doubt that introduction of complementary and family food leads to increased diversity of the gut microbiota , however, as reviewed below, the effect of introducing a more complex selection of microbial nutrients is probably a stronger driver of this development than the seeding of new microbes via food.
## Abiotic conditions
All the above mentioned sources, from which seeding occurs, have in common that in order for the microbes to be transmitted and establish in the gut, they have to be able to survive (i) in the source, (ii) during the transmission and (iii) in the gut environment. This means that although the gut environment is primarily anaerobic, the bacteria that grow there must typically have been able to survive exposure to oxygen during seeding, and are thus either facultative anaerobes (Enterobacteriales and Lactobacillales), anaerobes capable of surviving exposure to oxygen (Bifidobacteriales and Bacteroidales) or obligate but spore-forming anaerobes (some Clostridiales such as Clostridiaceae and Lachnospiraceae). An example of a curious exception to this is the strict anaerobic, non-spore forming Ruminococcaceae species F. prautznitzii, which, as described above, colonizes the infant gut quite late and its establishment in the gut seems to be accelerated by presence of siblings . It may be speculated that extensive exposure and close contact between individuals is necessary for the establishment of this organism. Interestingly, the presence of antioxidants such as cysteine and riboflavin in the local environment increases survival of F. prautznitzii during oxygen exposure [bib_ref] The gut anaerobe Faecalibacterium prausnitzii uses an extracellular electron shuttle to grow..., Khan [/bib_ref] [bib_ref] Antioxidants keep the potentially probiotic but highly oxygen-sensitive human gut bacterium Faecalibacterium..., Khan [/bib_ref] , suggesting that also the abiotic conditions in the environmental compartments may play a role for transmission.
After seeding (arrival of microorganisms into the gut) has taken place, the successional development of the intestinal microbiota during very early life is closely linked to changing abiotic conditions in this environment. This interaction is bidirectional since abiotic conditions, such as oxygen levels and pH, create distinct niches in which specific bacterial physiology types thrive and vice-versa that specific consortia of bacterial strains may alter these and other abiotic conditions over time. In addition to the abiotic factors, the immune system of the infant, as well as multiple factors present in breastmilk may play a role in shaping the infant microbiome. However, this is not within the scope of the current review.
## Oxygen
Several different gradients of partial oxygen pressure (pO 2 ) are present in the adult gastro-intestinal tract. These include a general longitudinal decrease from the proximal to the distal part [bib_ref] Noninvasive measurement of anatomic structure and intraluminal oxygenation in the gastrointestinal tract..., He [/bib_ref] , a very steep radial gradient from the intestinal submucosa to the lumen [bib_ref] Correlation between intraluminal oxygen gradient and radial partitioning of intestinal microbiota, Albenberg [/bib_ref] , which is most pronounced in the distal intestinal tract [bib_ref] Microbes vs. chemistry in the origin of the anaerobic gut lumen, Friedman [/bib_ref] and lastly a gradient along the crypt-villus axis in the small intestine due to counter-current blood flow [bib_ref] Evidence for the existence of a countercurrent exchanger in the small intestine..., Hallbäck [/bib_ref]. As a consequence of the radial pO 2 gradient, it was demonstrated in humans that a much lower ratio of obligate anaerobe to oxygen tolerant bacteria exists in rectal mucosal biopsies than in fecal samples [bib_ref] Correlation between intraluminal oxygen gradient and radial partitioning of intestinal microbiota, Albenberg [/bib_ref].
It is frequently reported that the neonatal intestine is relatively more oxygenated than later in life, thus initially only supporting growth of facultative anaerobic bacterial species, such as members of the Enterobacteriaceae family, and not obligate anaerobes. In line with this, it has been described that after the first week of life, the number and relative abundance of strictly anaerobic species found in a group of 25 infants increased with time [bib_ref] Mother-to-infant microbial transmission from different body sites shapes the developing infant gut..., Ferretti [/bib_ref]. The general belief is that the facultative anaerobes gradually reduce the oxygen levels and thus create the anaerobic environment of the developed gut [bib_ref] Dynamics and stabilization of the human gut microbiome during the first year..., Bäckhed [/bib_ref]. A recent study however challenged this notion, or at least narrow the timespan in which the intestinal lumen is oxygenated in early life . In a cohort of 88 healthy term infants sampled from 2 min to 176 h after birth it was indeed found that the facultative anaerobe E. coli was the most prevalent bacterium during the first 16 h of life. Several anaerobe species were however also abundant in some individuals, including B. vulgatus, B. dorei and Subdoligranulum spp. in this very early time-period, with quite some inter-individual heterogeneity at the species and strain level. Importantly, a mostly anaerobic environment was considered likely due to observed changes in serine, threonine and succinate levels in meconium samples consistent with anaerobic growth of E. coli. These findings indicate that the neonatal gut may be mostly anaerobic shortly after birth. The question thus remains whether it is possible that the neonatal intestinal lumen becomes anaerobic even in the absence of bacterial oxygen consumption, in spite of influx of oxygen from the endothelial cells.
The contribution of living bacteria versus purely chemical reactions to generate anaerobic conditions in the gut lumen has elegantly been explored recently in a germ-free mouse model [bib_ref] Microbes vs. chemistry in the origin of the anaerobic gut lumen, Friedman [/bib_ref] , revealing that the partial pressure of oxygen (pO 2 ) in the gut lumen is nearly identical in conventional and germ-free (GF) mice. Large longitudinal variations in pO 2 levels from stomach to cecum were found, however no difference was observed between germ-free and conventional animals. The highest levels of oxygen were recorded in the duodenum, which was attributed to the very large surface area allowing higher rates of diffusion of oxygen into the lumen as well as the low biomass causing limited microbial and chemical reduction of oxygen [bib_ref] Microbes vs. chemistry in the origin of the anaerobic gut lumen, Friedman [/bib_ref].
Chemical consumption of oxygen via lipid oxidation was demonstrated as an effective mechanism to reduce pO 2 levels in cecal content from GF animals albeit at much lower rates than observed using cecal content from conventional animals. In addition, the rate of oxygen consumption in GF cecal content was increased to a level similar to the conventional when spiked with facultative anaerobe E. coli, but not with the anaerobic but aerotolerant Clostridium sordelli [bib_ref] Oxygen tolerance of fresh clinical anaerobic bacteria, Tally [/bib_ref] [bib_ref] Microbes vs. chemistry in the origin of the anaerobic gut lumen, Friedman [/bib_ref]. These findings support the likely contribution of non-bacterial guided reduction in oxygen in the neonate gut, but also indicate that oxygen removal depends on the type of bacteria. Oppositely, increasing the luminal oxygenation by hyperbaric oxygen therapy in mice led to a decreased prevalence of catalase negative, obligate anaerobic Anaerostipes spp. (Albenberg et al. 2014).
In conclusion, although the neonatal colonic lumen probably rapidly becomes anaerobic, topographical differences in pO 2 are likely to exist and to affect gut microbiota composition in different compartments of the gut.
## Intestinal ph and transit time
Intestinal oxygen tension is closely connected with other physico-chemical factors including pH and redox potential. While the impact of pH on the differences between the microbial populations present in different sections of the GI tract is irrefutable, little is known about the impact of change in pH on the infant's intestinal microbial population.
The pH in the stomach of newborns at delivery is neutral [bib_ref] Gastric acidity in the first day of life, Avery [/bib_ref] , but fasting gastric pH in neonates rapidly drops to an average around 4.6 at age 2-6 days, and down to 2.6, which is similar to adults, already at 7-15 days of postnatal age [bib_ref] Continuous gastric pH measurement in young and older healthy preterm infants receiving..., Sondheimer [/bib_ref]. Studies assessing the pH in the gut of neonates are scarce, but one study, dated 1952, suggests that the luminal pH of the small intestine is lower in infants fed with cow's milk than in breastfed infants [bib_ref] Investigations on the bacterial flora, pH, and sugar content in the intestinal..., Barbero [/bib_ref]. The same study reveals that the pattern of small intestinal pH observed in breastfed infant aged 2 weeks to 3 months, with duodenal, jejunal and ileal pH around 6.4 ± 0.5, 6.6 ± 0.4 and 6.9 ± 0.5, is almost the same as that of older children and adults. Development of novel methodologies for non-invasive measurements of abiotic factors in the intestine is needed to obtain better knowledge about the processes occurring in the infant gastrointestinal tract. Such knowledge is highly relevant not only to understand the impact on intestinal microbes, but also to assess questions about solubility, absorption and metabolism of drugs and nutrients [bib_ref] Impact of regional differences along the gastrointestinal tract of healthy adults on..., Vertzoni [/bib_ref]. In vitro models of infant colonic microbial activity thus suggests that lactate metabolism is strongly modulated by abiotic factors such as pH and retention time [bib_ref] Lactate metabolism is strongly modulated by fecal inoculum, pH, and retention time..., Pham [/bib_ref].
In adults, colonic pH plays a key role in determining the outcome of microbial interspecies competition and response to dietary fibers [bib_ref] Modulation of the human gut microbiota by dietary fibres occurs at the..., Chung [/bib_ref] as well as in amino acid metabolism [bib_ref] Enumeration of human colonic bacteria producing phenolic and indolic compounds: effects of..., Smith [/bib_ref]. In example, whereas propionate-producing Bacteroides spp. dominate fecal microbial communities cultured at pH > 6.0, butyrate-producing Eubacterium/Roseburia or Faecalibacterium spp. dominate when pH is lowered to 5.5 [bib_ref] pH and peptide supply can radically alter bacterial populations and short-chain fatty..., Walker [/bib_ref] [bib_ref] Modulation of the human gut microbiota by dietary fibres occurs at the..., Chung [/bib_ref] , but the species that are dominating depends on the diet (e.g. type of dietary fiber and peptides). Opposite the small intestinal luminal pH [bib_ref] Microbes vs. chemistry in the origin of the anaerobic gut lumen, Friedman [/bib_ref] , the fecal pH is significantly lower in breastfed compared to formula fed infants [bib_ref] Volatile fatty acids, lactic acid, and pH in the stools of breast-fed..., Ogawa [/bib_ref] [bib_ref] Effect of a fermented formula on thymus size and stool pH in..., Indrio [/bib_ref]. The low fecal pH in breastfed infants is maintained by human milk oligosaccharide (HMO) degrading, acetate-producing Bifidobacterium species [bib_ref] A key genetic factor for fucosyllactose utilization affects infant gut microbiota development, Matsuki [/bib_ref] [bib_ref] Elevated fecal pH indicates a profound change in the breastfed infant gut..., Henrick [/bib_ref] , and this is likely to prevent or reduce colonization of non-acid tolerant bacteria. Formula fed infants are not dominated by Bifidobacterium spp. [bib_ref] Intestinal microbiota of 6-weekold infants across Europe: geographic influence beyond delivery mode,..., Fallani [/bib_ref] , and have a much more diverse microbiota [bib_ref] Temporal development of the gut microbiome in early childhood from the TEDDY..., Stewart [/bib_ref] , possibly due to the more permissive pH conditions in the colonic environment. Colonic pH is additionally known to be affected by intestinal transit time [bib_ref] Increasing butyrate concentration in the distal colon by accelerating intestinal transit, Lewis [/bib_ref] , and recently, we have reported that in adults, transit time has a major impact on microbiome diversity and composition as well as on microbial metabolism [bib_ref] Colonic transit time is related to bacterial metabolism and mucosal turnover in..., Roager [/bib_ref]. Although it remains to be investigated, we speculate that this is the case also in the infant gut.
## Effects of antibiotic treatment on abiotic conditions in the infant gut
It is well known from animal studies that oral intake of antibiotics courses acute and profound changes in bacterial community composition, which is determined by the dose and class of antibiotic [bib_ref] Antibiotic treatment affects intestinal permeability and gut microbial composition in wistar rats..., Tulstrup [/bib_ref]. Lately, the effects of antibiotics on microbial communities in early life and health risks associated with these changes have been investigated in depth and several comprehensive reviews are available [bib_ref] Antibiotics in early life and obesity, Cox [/bib_ref] [bib_ref] Risks of antibiotic exposures early in life on the developing microbiome, Schulfer [/bib_ref]. Importantly, antibiotic-induced early life disturbances have been associated with increased risk of developing asthma [bib_ref] Association of infant antibiotic exposure with childhood health outcomes, Aversa [/bib_ref] and may also have lasting metabolic consequences [bib_ref] Altering the intestinal microbiota during a critical developmental window has lasting metabolic..., Cox [/bib_ref]. Here, we focus on the putative effects of antibiotic use on the abiotic conditions in the intestine.
In the adult colon, there is a steep gradient from the oxygenated endothelial cells into the lumen (Espey 2013). Colonocytes oxidize microbially-produced butyrate as their preferred carbon source, thereby consuming oxygen and generating ATP (Rivera-Chávez, Lopez and Bäumler 2017). Orally administrated antibiotics are known to cause an increase in intestinal oxygen levels, which may result in an expansion of facultative anaerobic bacteria within the Enterobacteriaceae family [bib_ref] Depletion of butyrateproducing clostridia from the gut microbiota drives an aerobic luminal..., Rivera-Chávez [/bib_ref]. This antibiotic-induced increase in oxygen is partly explained by lack of butyrate production by Clostridia (Rivera-Chávez, Lopez and Bäumler 2017). Antibiotics-induced blooms in Enterobacteriaceae are however also commonly observed during early infancy [bib_ref] Influence of antibiotic exposure in the early postnatal period on the development..., Tanaka [/bib_ref] [bib_ref] High-throughput sequencing reveals the incomplete, short-term recovery of infant gut microbiota following..., Fouhy [/bib_ref] , although butyrate producing bacteria are only present at very low levels at this age [bib_ref] Initial butyrate producers during infant gut microbiota development are endospore formers, Appert [/bib_ref]. This might be explained by induction of nitrate production in the gut. It has thus been shown in mice that streptomycin treatment induces gut mucosal nos2 expression, which increases luminal concentrations of nitrate that functions as an alternative electron acceptor for E. coli and promotes blooming of this species [bib_ref] Streptomycin-induced inflammation enhances Escherichia coli gut colonization through nitrate respiration, Spees [/bib_ref].
An increase in the oxygen/nitrate levels in the intestine would also manifest as an increase in redox potential, which defines the overall environmental capacity for reducing chemical reactions and is thus a key metric to describe the state of the microbial community [bib_ref] Antibiotic-induced changes in the microbiota disrupt redox dynamics in the gut, Reese [/bib_ref]. It has recently been suggested that availability of electron acceptors (redox potential) is an important factor driving the structure of bacterial communities, and Enterobacteriaceae levels correlate positively with redox potential [bib_ref] Antibiotic-induced changes in the microbiota disrupt redox dynamics in the gut, Reese [/bib_ref]. An increase in the redox potential, characterized by a higher overall oxidant to reductant ratio, may thus result in opportunistic pathogens taking advantage of new respiratory pathways and is additionally known to increase oxidative stress due to generation of free radicals and ROS [bib_ref] Free radicals, reactive oxygen species, oxidative stress and its classification, Lushchak [/bib_ref].
In order to understand and predict the effects of antibiotic treatment in the infant gut, it is therefore important not only to take into account the effect of a given antibiotic on the targetand non-target bacteria, but also to consider both the direct effects on host gastrointestinal cells and the consequences of removal of non-target bacteria for the abiotic intestinal environment, which to a large degree shapes the microbiota.
## Nutrient availability, competition and cross feeding
The intestinal environment is characterized by a continuous turnover of the bacterial population through proliferation and fecal excretion, combined with a continuous supply of nutrients.
With respect to these features, the intestine is thus comparable to a chemostat or continuous flow culture. This was extensively investigated and discussed in a series of papers already in 1983 . An important conclusion derived from this perspective is that the number of bacteria that can stably coexist in a chemostat, is determined by the number of different available nutrients. Additionally, in a flow-culture, the populations of bacteria are controlled by one or a few nutritional substrates which a given strain can utilize most efficiently under the prevailing abiotic conditions [bib_ref] Mechanisms that control bacterial populations in continuous-flow culture models of mouse large..., Freter [/bib_ref].
Translated to the intestinal microbial ecosystem, this means that an increase in the complexity of bacterial nutrients, which in the gut originate from ingested diet as well as from mucosal turnover, will drive an increase in the diversity of the bacterial community. In line with this, it was recently demonstrated that dietary diversity scores were positively correlated with microbial diversity in infant stool samples (109). Also in a large adult population (N > 1500 samples), it was found that a higher number of different types of vegetables in the diet was associated with a higher alpha diversity of the fecal bacterial population [bib_ref] American gut: an open platform for citizen science microbiome research, Mcdonald [/bib_ref]. Furthermore, it has been elegantly demonstrated in mice, that the abundance of a given strain can be regulated by changing the concentration of a substrate exclusively accessible by this strain [bib_ref] An exclusive metabolic niche enables strain engraftment in the gut microbiota, Shepherd [/bib_ref]. These relations between dietary complexity, nutrient utilization capacity and shaping of a bacterial community become very clear from studies of the development of the infant gut microbiota, as reviewed in the sections below.
## Breastfeeding
After lactose and fat, HMOs constitute the third most abundant solid component of human breastmilk [bib_ref] Human Milk Oligosaccharides (HMOS): structure, function, and enzyme-catalyzed synthesis, Chen [/bib_ref]. While lactose and fat can be digested by the infant's digestive enzymes, and thus to a lesser degree reach the intestinal bacterial communities, the HMOs are indigestible to the human host, and thus available to the gut bacteria in very high amounts. A multitude of studies show that the gut of breastfed infants (as opposed to formula-fed infants) is dominated by the specific bacterial species, which are able to digest HMOs [bib_ref] Temporal development of the gut microbiome in early childhood from the TEDDY..., Stewart [/bib_ref]. These include specific species of Bifidobacterium such as . For some of these species, it is not only the production of specific enzymes capable of HMO degradation, which makes them highly competitive on this substrate, but also their expression of membrane transporters with high affinity for the different types of oligosaccharides present in breastmilk . Thus, while B. bifidum employs extracellular enzymes (fucosidases, sialidases and lacto-N-biosidases) to degrade HMOs and transport only the released di-and monosaccharides into the cytoplasm, B. longum subsp. infantis internalizes the intact HMO structures via highly specific membrane transporters and subsequently degrade the HMOs intracellularly . In addition to the specialized infant bifidobacteria, also species of Bacteroides have been reported to be able to degrade HMOs by use of pathways developed for mucus-utilization [bib_ref] Bacteroides in the infant gut consume milk oligosaccharides via mucusutilization pathways, Marcobal [/bib_ref] , as discussed below. It was moreover recently demonstrated that Roseburia and Eubacterium species utilize selected HMOs as well as HMO and mucin degradants [bib_ref] Butyrate producing colonic clostridiales metabolise human milk oligosaccharides and cross feed on..., Pichler [/bib_ref]. Combined with the ability of these species to utilize plant derived carbohydrates such as xylans and β-mannans [bib_ref] Dietary fibre degradation and fermentation by two xylanolytic bacteria Bacteroides xylanisolvens XB1A..., Mirande [/bib_ref] [bib_ref] Differential bacterial capture and transport preferences facilitate co-growth on dietary xylan in..., Leth [/bib_ref] [bib_ref] The human gut Firmicute Roseburia intestinalis is a primary degrader of dietary..., Rosa [/bib_ref] , this provides a plausible explanation for their appearance in the infant gut during weaning [bib_ref] Determinants of the human infant intestinal microbiota after the introduction of first..., Fallani [/bib_ref]. Nevertheless, the fact that HMOs are highly selective and constitute a considerable amount of the bacterial nutrients in breastfed infants is underlined by the observation that cessation of breastfeeding has a larger impact on microbiota development than introduction of solid foods [bib_ref] Temporal development of the gut microbiome in early childhood from the TEDDY..., Stewart [/bib_ref].
## Bacterial nutrients in infant and child food
In many countries, iron fortification of infant formula or iron supplementation given as droplets for infants are recommended in order to prevent iron deficiency. Iron availability may well be a growth-limiting factor for specific bacteria inhabiting the infant intestine [bib_ref] Excess iron intake as a factor in growth, infections, and development of..., Lönnerdal [/bib_ref] , and iron supplementation may thus have an impact on the developing microbiota. However, as recently reviewed by others , the currently available reports about effects of iron supplementation on the intestinal microbial composition in infants are not in consensus.
Longitudinal observational studies have identified associations between toddler diet and gut microbiota [bib_ref] Dietary intake influences gut microbiota development of healthy Australian children from the..., Matsuyama [/bib_ref]. As the child starts to consume more and more different types of food items, the gradual transition to 'adult' food, and an increased intake of protein as well as fibre is associated with a gradual increase of intestinal microbial diversity and of the abundance of Lachnospiraceae [bib_ref] Infant gut microbiota development is Driven by transition to family foods independent..., Laursen [/bib_ref]. Given that the accessibility of amino acids is not a limiting factor for gut bacteria [bib_ref] Glyphosate has limited short-term effects on commensal bacterial community composition in the..., Nielsen [/bib_ref] , the bacterial carbohydrate sources (fibres, polysaccharides), are likely to be major drivers not only of diversity, but also of the composition of the bacterial community, since they are selective for the specific bacteria that have the capacity to digest them [bib_ref] Microbial degradation of complex carbohydrates in the gut, Flint [/bib_ref]. Bacterial phyla that are highly specialized in carbohydrate utilization, as revealed by the percentage of genes on their genome dedicated to this task, are the Bacteriodetes and the Actinobacteria [bib_ref] Nursing our microbiota: molecular linkages between bifidobacteria and milk oligosaccharides, Sela [/bib_ref]. Noteworthy, of these two phyla, the Actinobacteria have dedicated a larger part of their genome also to transport of oligo-and polysaccharides into the cytoplasm. The genera Bifidobacterium and Bacteroides within these phyla, along with the Roseburia-Eubacterium group, all contain species that can use both HMOs and complex plant polysaccharides as carbohydrate sources, and consistently these taxa are dominating in the transition phase between breastfeeding and family diet [bib_ref] Temporal development of the gut microbiome in early childhood from the TEDDY..., Stewart [/bib_ref]. Multiple reports from in vitro studies [bib_ref] Feruloylated and nonferuloylated arabino-oligosaccharides from sugar beet pectin selectively stimulate the growth..., Holck [/bib_ref] , animal studies [bib_ref] An exclusive metabolic niche enables strain engraftment in the gut microbiota, Shepherd [/bib_ref] and human trials [bib_ref] A low-gluten diet induces changes in the intestinal microbiome of healthy Danish..., Hansen [/bib_ref] reveal that the specific features of the dietary polysaccharides (nature of the monomers, binding structures, branching) are selectively promoting specific bacterial species and subspecies. The composition of dietary polysaccharides are thus likely to shape the development of the child's microbiota during transition from breastfeeding and baby food to other types of solid complementary feeding.
## Host mucins as bacterial nutrients
The complexity of the nutritional landscape encountered by gut bacteria is increased by the fact that the gut contains several sub-systems, offering different types of niches as the encountered conditions are different for bacteria residing in the mucus layer, in the lumen, in the small intestine and in the large intestine [bib_ref] Microbial nutrient niches in the gut, Pereira [/bib_ref]. Although interconnected, these spatial niches govern competitive advantages in distinct directions [bib_ref] Physiological state of Escherichia coli BJ4 growing in the large intestines of..., Poulsen [/bib_ref]. In humans, as faecal samples are often the object of study, such niche-specific competitive interactions are typically not possible to measure, and knowledge about their impact in development of the bacterial community is scarce.
The intestinal mucus is composed of a firmly adherent layer, close to the epithelial cells, which typically does not contain many bacteria, and a loosely adherent layer, which is extensively colonized by a variety of microbes [bib_ref] The adherent gastrointestinal mucus gel layer: thickness and physical state in vivo, Atuma [/bib_ref]. The microbiota of the mucus layer in adults is known to differ from that found in fecal samples [bib_ref] Mucosaassociated bacteria in the human gastrointestinal tract are uniformly distributed along the..., Zoetendal [/bib_ref]. In 2004, a new species, Akkermansia muciniphila, was described, which specializes in utilizing intestinal mucin as the only carbon-and nitrogen source and grew rather poorly on alternative sugars [bib_ref] Akkermansia municiphila gen. nov., sp. nov., a human intestinal mucindegrading bacterium, Derrien [/bib_ref]. A. muciniphila has since then been extensively studied, and it is found primarily in the human colon, but also in breastmilk. It is found only in low levels in the infant intestine, but during the first years of life, its abundance reaches that found in adults [bib_ref] Intestinal integrity and Akkermansia muciniphila, a mucin-degrading member of the intestinal microbiota..., Collado [/bib_ref] , suggesting that the development of the intestinal environment supports the colonization of this bacterium.
Not only Akkermansia, but also gut bacteria belonging to the genera Bacteroides (e.g. Bacteroides thetaiotaomicron and Bacteroides fragilis), Ruminococcus (e.g. Ruminococcus gnavus) and Bifidobacterium (e.g. B. bifidum) are able to degrade glycans from intestinal mucins, although the latter only with moderate efficiency [bib_ref] Mucin glycan foraging in the human gut microbiome, Tailford [/bib_ref]. Unlike A. muciniphila, these species abundantly colonize the infant gut early and are capable of utilization of HMOs from breast milk and dietary complex polysaccharides as well as mucins, and thus contain the metabolic flexibility to also become very robust and consistent members of the adult microbiota. Particularly, B. thetaiotaomicron is well studied with respect to its ability to switch between the complex carbohydrate sources offered by diet and host mucins, respectively [bib_ref] Glycan foraging in vivo by an intestine-adapted bacterial symbiont, Sonnenburg [/bib_ref] , and is a highly abundant and prevalent member of the adult gut microbiota.
## Bacterial cross feeding
Trophic interactions occur between members of the infant gut microbiota. A well-studied example is interspecies crossfeeding within the genus Bifidobacterium, in which different strains can display an 'altruistic' as well as a 'selfish' type of HMO metabolism as described in the following.
By employing extracellular fucosidases and sialidases when grown on HMOs or mucins, B. bifidum can liberate fucose and sialic acid, which B. breve can internalize and metabolize [bib_ref] Bifidobacterium bifidum ATCC 15696 and Bifidobacterium breve 24b metabolic interaction based on..., Centanni [/bib_ref]. A similar growth enhancing effect of B. bifidum on B. longum subs. longum has been observed when co-cultured in presence of HMOs [bib_ref] Sharing of human milk oligosaccharides degradants within bifidobacterial communities in faecal cultures..., Gotoh [/bib_ref]. Indeed, in vitro experiments have demonstrated an increased abundance of other Bifidobacterium species when human fecal cultures incubated with HMOs are spiked with B. bifidum, underlining the 'altruistic' activity of this species [bib_ref] Sharing of human milk oligosaccharides degradants within bifidobacterial communities in faecal cultures..., Gotoh [/bib_ref].
Also some isolates of B. kashiwanohense express fucosidases that liberates fucose in the culture medium when grown on the HMOs 2 FL (2 fucosyllactose) or 3 FL(3 fucosyllactose) [bib_ref] Fucosyllactose and L-fucose utilization of infant Bifidobacterium longum and Bifidobacterium kashiwanohense, Bunesova [/bib_ref] [bib_ref] Metabolism of the predominant human milk oligosaccharide fucosyllactose by an infant gut..., James [/bib_ref] , and may cross feed to fucose-utilizers such as B. breve [bib_ref] Trophic interactions of infant bifidobacteria and eubacterium hallii during L-fucose and fucosyllactose..., Schwab [/bib_ref].
Infant isolates of B. pseudocatenulatum grow on HMOs [bib_ref] A key genetic factor for fucosyllactose utilization affects infant gut microbiota development, Matsuki [/bib_ref] , and HMO-degrading B. pseudocatenulatum strains have been shown to support growth of a non-HMO utilizing B. longum subsp. longum strain isolated from the same infant [bib_ref] Breast milkderived human milk oligosaccharides promote Bifidobacterium interactions within a single ecosystem, Lawson [/bib_ref]. The opposite may also occur, since when strains isolated from another infant were observed, a HMOdegrading B. longum subsp. longum strain supported growth of non-HMO utilizing B. pseudocatenulatum strains. Fucose, galactose, acetate and N-acetylglucosamine were identified as key by-products of bifidobacterial degradation/metabolism of HMOs that mediate interspecies cross-feeding [bib_ref] Breast milkderived human milk oligosaccharides promote Bifidobacterium interactions within a single ecosystem, Lawson [/bib_ref]. Thus, cross feeding between Bifidobacterium species is likely to explain the co-occurrence of different Bifidobacterium species commonly observed in the infant gut [bib_ref] Diversity of bifidobacteria within the infant gut microbiota, Turroni [/bib_ref] [bib_ref] Glycan utilization and crossfeeding activities by bifidobacteria, Turroni [/bib_ref].
In contrast to the 'altruistic' behavior displayed by B. bifidum and other specific bifidobacterial strains, B. longum subsp. infantis strains often display a 'selfish' HMO metabolism, internalising the intact HMO structures prior to degradation, and thereby leaving no HMO remnants for others to consume [bib_ref] Breast milkderived human milk oligosaccharides promote Bifidobacterium interactions within a single ecosystem, Lawson [/bib_ref]. Consequently, the microbiota if breastfed infants colonized with efficient HMO-utilizing strains of B. longum subsp. Infantis is often completely dominated by this strain, .
Growing on HMO or mucins in the infant intestine, Bifidobacterium species can also cross feed with strains belonging to other genera, due to their release of simple sugar constituents such as lactose, galactose and N-acetyl glucosamine, and products of bifidobacterial metabolism such as acetate, lactate and 1,2-propanediol, which support growth e.g. of Eubacterium hallii [bib_ref] Trophic interactions of infant bifidobacteria and eubacterium hallii during L-fucose and fucosyllactose..., Schwab [/bib_ref] [bib_ref] Mucin cross-feeding of infant Bifidobacteria and Eubacterium hallii, Bunesova [/bib_ref].
This results in the formation of butyrate and propionate, which increase in abundance during complementary feeding [bib_ref] Timing of complementary feeding is associated with gut microbiota diversity and composition..., Differding [/bib_ref].
Indeed, plant derived substrates such as inulin/oligofructose and arabinoxylan oligosaccharides introduced with complementary feeding have been identified as bifidogenic, but also promote the growth of various butyrate producing bacteria including Eubacterium rectale, Roseburia spp. and F. prausnitzii because Bifidobacterium-produced acetate is utilized by the butyrate producers [bib_ref] Cross-feeding between Bifidobacterium longum BB536 and acetate-converting, butyrate-producing colon bacteria during growth..., Falony [/bib_ref] [bib_ref] Mutual cross-feeding interactions between Bifidobacterium longum subsp. longum NCC2705 and Eubacterium rectale..., Rivière [/bib_ref] [bib_ref] Bifidobacterial inulin-type fructan degradation capacity determines cross-feeding interactions between bifidobacteria and Faecalibacterium..., Moens [/bib_ref].
It has been demonstrated that cross-feeding on arabinoxylan oligosaccharides is mutually beneficial between B. longum subsp. longum and E. rectale. B. longum uses the arabinose part to produce acetate and xylo-oligosaccharides, and from these xylooligosaccharides E. rectale releases xylose monomers, which are substrates for B. longum [bib_ref] Mutual cross-feeding interactions between Bifidobacterium longum subsp. longum NCC2705 and Eubacterium rectale..., Rivière [/bib_ref]. Thus, it is likely that trophic mutualism between bifidobacteria and butyrate producing taxa exists in the infant gut during complementary feeding.
It has been speculated that lactate cross-feeding and conversion into short chain fatty acids is a key to ecosystem stability in the adult human gut [bib_ref] Pivotal roles for pH, lactate, and lactate-utilizing bacteria in the stability of..., Wang [/bib_ref]. Lactate is however also a common metabolic end-product of many early life gut colonizers. Lactate-producing bacteria such as Lactobacillus, Streptococcus, Staphylococcus, Bacteroides and Bifidobacterium may support the growth of lactate consuming bacteria including Veillonella and E. hallii [bib_ref] Early colonization of functional groups of microbes in the infant gut, Pham [/bib_ref]. In example, B. bifidum produces lactate, which is consumed by co-cultivated E. hallii [bib_ref] Mucin cross-feeding of infant Bifidobacteria and Eubacterium hallii, Bunesova [/bib_ref]. Additionally, a correlation is reported between the lactate consuming Cutibacterium avidum (formerly Propionibacterium), and the lactate producers Bifidobacterium and Streptococcus in infant feces [bib_ref] Colonization of Cutibacterium avidum during infant gut microbiota establishment, Rocha Martin [/bib_ref].
In summary, consumption of degradation products (diet or mucin derived mono-and di-saccharides) and/or metabolites (such as lactate, acetate and 1,2-propanediol) from lactic acid bacteria and bifidobacteria is thus likely to allow propionate and butyrate producers to establish in the infant gut and promote maturation into a diverse and stable adult-like gut microbial community.
## Ecological aspects
It is well established, that the developing infant microbiota undergoes an increase in within-individual diversity (alpha diversity), and a decrease in between-individual diversity (beta diversity; [bib_ref] The intestinal microbiome in early life: health and disease, Arrieta [/bib_ref] [bib_ref] Establishment of intestinal microbiota during early life: a longitudinal, explorative study of..., Bergström [/bib_ref]. In the gut of a newborn infant, where microorganisms are absent or very scarce, the seeding of new microorganisms from the environment will have a major influence on microbiota composition, whereas the impact of competition will increase as the community increases in density and diversity .
An new ecological aspect originates from a recent study describing the healthy or impaired development of the infant gut microbiota based on a co-varying set of key microbial taxa, a so-called ecogroup [bib_ref] A sparse covarying unit that describes healthy and impaired human gut microbiota..., Raman [/bib_ref]. This approach takes longitudinal microbial interactions or networks into account in the description of developing microbial communities. An ecogroup of 15 bacterial taxa was found to explain the majority of variation in the gut microbiome and consistently co-varied during the first 5 years of life in cohorts of Bangladeshi, Peruvian or Indian children, respectively. Importantly, ecogroup taxa configurations were found to be altered in malnourished infants. The study highlights the utility of co-variation networks applied to longitudinal data in order to increase our understanding of the ecological patterns of the developing gut microbiota.
In order to understand the putative impact of order of exposure to microorganisms originating from the sources of seeding discussed above, it is useful to consider the fundamental mechanisms governing competition for ecological niches. It has been suggested that predictions about impact of order of seeding on community assembly (in any ecosystem) can be based on considering the following three features of the species' ecological niches: Overlap, impact and requirement [bib_ref] Historical contingency in species interactions: towards niche-based predictions, Vannette [/bib_ref] [bib_ref] Role of priority effects in the early-life assembly of the gut microbiota, Sprockett [/bib_ref]. A so-called 'priority effect' designates the observation that a given species gains an advantage or a disadvantage by arriving to an ecosystem earlier than others. If two species are competing for the same niche (overlap), the priority effect will be strong. This is speculated to be the case e.g. when specialized HMO consumers compete for this nutritional niche. It has been elegantly demonstrated in a neonatal mouse model, that for the vast majority of intestinal bacteria, early arrival represents an advantage [bib_ref] Experimental evaluation of the importance of colonization history in early-life gut microbiota..., Martínez [/bib_ref]. However, it was also observed that in some cases, establishment in the neonatal mouse gut was facilitated by later arrival, suggesting that the preexisting microcoorganisms may impact on the ecological niched either to facilitate or to prevent the establishment of given newcomers, as when facultative organisms consume the intestinal oxygen and thereby facilitates establishment of anaerobic species, but prevent proliferation of aerobes seeded into the gut. Finally, species that are more sensitive to specific requirements, i.e. whose growth rate is sensitive to abiotic and nutritional environmental changes, are predicted to experience stronger priority effects [bib_ref] Historical contingency in species interactions: towards niche-based predictions, Vannette [/bib_ref] [bib_ref] Role of priority effects in the early-life assembly of the gut microbiota, Sprockett [/bib_ref]. This suggests that species such as B. thetaiotaomicron, which have many options for nutritional sources, can robustly colonize the infant intestine irrespective of order of exposure.
Since closely related species might be more likely to compete for overlapping niches [bib_ref] The merging of community ecology and phylogenetic biology, Cavender-Bares [/bib_ref] , it could be speculated that the effect of early arrival would be most pronounced on the competition between bacteria that are phylogenetically closely related, however this was contradicted by experiments in mice [bib_ref] Experimental evaluation of the importance of colonization history in early-life gut microbiota..., Martínez [/bib_ref] , suggesting that the bacterial functions and features governing competition for niches are not necessarily related to phylogeny.
In addition to investigate the successional development of the neonatal intestinal microbiota based on taxa [bib_ref] Dynamics and stabilization of the human gut microbiome during the first year..., Bäckhed [/bib_ref] it is also possible to study the succession of bacterial traits (phenotypes), an approach well developed in environmental ecology [bib_ref] A trait-based approach to community assembly: partitioning of species trait values into..., Ackerly [/bib_ref]. Recently, a study of trait-based community assembly of the microbiota in a cohort of 56 infants during the first 3 years of life (Guittar, Shade and Litchman 2019) elucidated some important general features. First, the study demonstrates that the average 16S rRNA gene copy number per bacterial genome decreases as a function of time during the first 2-3 years of life. Since a high number of ribosomal gene copies is affiliated to the ability of rapid initiation of protein synthesis and thus rapid response to conditions allowing growth [bib_ref] rRNA operon copy number reflects ecological strategies of bacteria, Klappenbach [/bib_ref] , this suggests that the infant gut undergoes a transition from initially fast responding strains to the later predominance of overall less rapidly adapting and slower growing species. Indeed, it has been observed in mice that the in situ ribosome content of E. coli growing in intestinal mucus reflects rapid growth [bib_ref] Physiological state of Escherichia coli BJ4 growing in the large intestines of..., Poulsen [/bib_ref]. We thus speculate than in the neonatal intestine, rapid response to changing conditions and capacity for rapid proliferation are selective factors for initial establishment. In support of this, an acute surge in total bacterial density quantified by 16S rRNA gene copies per gram intestinal content has been observed as a consequence of antibiotic treatment of rats, and could partly be explained by a switch to more rapidly adapting bacterial species [bib_ref] Antibiotic treatment affects intestinal permeability and gut microbial composition in wistar rats..., Tulstrup [/bib_ref].
The observed temporal decrease in rRNA gene copies per genome in the developing gut microbiota was accompanied by a concurrent decrease in predicted motility scores and oxygen tolerance from 3 to 9 months, as well as by increased temperature optimum and increased sporulation capacity in the same period [bib_ref] Trait-based community assembly and succession of the infant gut microbiome, Guittar [/bib_ref] , likely reflecting the impact of these traits at different periods of infancy.
## Concluding remarks
The first 1000 days after birth of an infant, are often referred to as a window of opportunity for shaping the microbiota, which will characterize the individual throughout life [bib_ref] The composition of the gut microbiota throughout life, with an emphasis on..., Rodríguez [/bib_ref]. Since this period is also very important for development of the immune system, where exposure to microbes play a pivotal role [bib_ref] How colonization by microbiota in early life shapes the immune system, Gensollen [/bib_ref] , the processes governing the microbiota assembly are important to elucidate.
Here, we have taken the perspective of microbial physiology and ecology to describe the development of the microbiota. In the first period after birth, the bacterial load in the intestine is low, meaning that many ecological niches are free for the seeded bacteria to explore. The high inter-individual (beta) diversity at this stage is probably reflecting the many different sources of seeding to the gut at this stage.
However, proliferation of seeded bacteria of a given species requires abiotic conditions that allow for this, which is reflected in the observation that vaginal and skin-derived species remain only transiently in the infant gut, while maternal gut species represent a significant part of the establishing microbiota as reviewed above.
As the bacterial load and diversity increases, competition for nutritional niches originating from diet and mucosa will play a larger and larger role . As long as the nutritional environment is governed by breastfeeding, bacteria that are efficient in using the HMOs dominate the community. When the complexity of diet increases, so does the complexity (alpha diversity) of the bacterial community, and eventually the impact of original seeding sources is no longer detectable. Additionally, the increasing hostility of the gut environment, characterized by reduced oxygen availability and reduced pH and later by scarceness of bacterial nutrients subject to competition, selects for a community that is optimized for coping with this, thereby reducing the beta diversity.
In order to better understand and predict this development, and eventually to decipher how to govern and optimize it to improve health, we find that it is pivotal to combine longitudinal infant studies with fundamental studies of bacterial growth physiology as well as with ecological models.
ACKNOWLEDGMENTSThe authors thank Mikiyasu Sakanaka for useful input about glycan utilization in bifidobacteria. |
Targeting prodromal Alzheimer’s disease: too late for prevention?
[bib_ref] Synaptic proteins and phospholipids are increased in gerbil brain by administering uridine..., Wurtman [/bib_ref] [bib_ref] Efficacy of a medical food in mild Alzheimer's disease: a randomized, controlled..., Scheltens [/bib_ref] [bib_ref] Efficacy of Souvenaid in mild Alzheimer's disease: results from a randomized, controlled..., Scheltens [/bib_ref] [bib_ref] The S-Connect study: results from a randomized, controlled trial of Souvenaid in..., Shah [/bib_ref]
## Hhs public access
Several important lessons can be learned from the LipiDiDiet trial. First, LipiDiDiet, together with past trials, does not provide sufficient evidence for the use of Fortasyn Connect in mild or prodromal Alzheimer's disease. The suggestion of benefit in two of the secondary outcomes is encouraging, but needs to be confirmed in further research.
Second, most of the participants who were screened were included in the trial, which was attributed to participants primarily coming from memory clinic populations with recent detailed assessments. Although this approach enhanced the selection of this group of individuals, it limits the external validity of this study outside of specialised memory clinics.
Third, baseline memory scores were very similar to mild Alzheimer's disease, with a higherthan-expected conversion rate to Alzheimer's disease during the trial. Despite this extent of impairment, changes in the primary outcome were not significant. This result indicates the limitations and challenges of constructing composite outcomes in the absence of validated data relevant to the population studied.
Fourth, the intervention appears to be targeting the disease at a late stage. A preplanned analysis in LipiDiDiet suggested that better baseline cognitive function (Mini-Mental State Examination score ≥26) was associated with a better drug placebo response in CDR-SB, hippocampal volume, and the primary endpoint in the per-protocol analysis.
Prodromal Alzheimer's disease defined by the IWG-1 criteria 2 requires evidence of neurodegeneration, such as medial temporal lobe atrophy, for inclusion into the study. As neurodegeneration progresses, the ability of preventive therapies to reverse progression to Alzheimer's disease is attenuated. [bib_ref] Association of docosahexaenoic acid supplementation with Alzheimer disease stage in apolipoprotein E..., Yassine [/bib_ref] Identification of people at risk of Alzheimer's disease, but who have no evidence of significant neurodegeneration, is crucial to the success of preventive strategies. Sensitive and less invasive biomarkers are needed to correctly identify Alzheimer's disease at its earliest stages for long-term prevention interventions. |
Establishing a health demographic surveillance site in Bhaktapur district, Nepal: initial experiences and findings
Background: A health demographic surveillance system (HDSS) provides longitudinal data regarding health and demography in countries with coverage error and poor quality data on vital registration systems due to lack of public awareness, inadequate legal basis and limited use of data in health planning. The health system in Nepal, a low-income country, does not focus primarily on health registration, and does not conduct regular health data collection. This study aimed to initiate and establish the first HDSS in Nepal. Results: We conducted a baseline survey in Jhaukhel and Duwakot, two villages in Bhaktapur district. The study surveyed 2,712 households comprising a total population of 13,669. The sex ratio in the study area was 101 males per 100 females and the average household size was 5. The crude birth and death rates were 9.7 and 3.9/1,000 population/year, respectively. About 11% of births occurred at home, and we found no mortality in infants and children less than 5 years of age. Various health problems were found commonly and some of them include respiratory problems (41.9%); headache, vertigo and dizziness (16.7%); bone and joint pain (14.4%); gastrointestinal problems (13.9%); heart disease, including hypertension (8.8%); accidents and injuries (2.9%); and diabetes mellitus (2.6%). The prevalence of non-communicable disease (NCD) was 4.3% (95% CI: 3.83; 4.86) among individuals older than 30 years. Age-adjusted odds ratios showed that risk factors, such as sex, ethnic group, occupation and education, associated with NCD.Conclusion: Our baseline survey demonstrated that it is possible to collect accurate and reliable data in a village setting in Nepal, and this study successfully established an HDSS site. We determined that both maternal and child health are better in the surveillance site compared to the entire country. Risk factors associated with NCDs dominated morbidity and mortality patterns.
# Background
The current estimation of disease burden in Nepal is based on cross-sectional studies, including the National Census and studies conducted by the Ministry of Health and other agencies. These health data lack complete epidemiological information to support critical decisions by health planners, policymakers and health managers. Additionally, Nepal's registration systems for vital statistics include coverage errors (e.g., only 35% of live births are registered and no causes of deaths were available) due to lack of public awareness, inadequate legal basis and limited use in health planning and policy [bib_ref] New Delhi: WHO Regional Office for South East Asia: SEA-HSD-304, Gautam [/bib_ref]. Proper planning and budget allocations for appropriate preventive measures require reliable, accurate and continuous information about community-level health issues [bib_ref] Epidemiological approaches to health planning, management and evaluation, Woodall [/bib_ref] [bib_ref] Epidemiology's contribution to health service management and planning in developing countries: a..., Unger [/bib_ref].
A Health Demographic Surveillance System (HDSS) provides a method for collecting data, based on longitudinal measurement of population dynamics, health and social changes, to inform and advocate public health policymakers and practitioners [bib_ref] Research into health, population and social transitions in rural South Africa: Data..., Kahn [/bib_ref]. Such surveillance sites are restricted to a specified geographic area, and data are collected at regular intervals. Many low-and middle-income countries in Asia and Africa have already established HDSS under INDEPTH, an international network of field sites, thus providing continuous demographic evaluation of population and health in developing countries . By undertaking population-level research, HDSS also engender a thorough understanding of disease burden. Using interventional studies, HDSS can also be useful in studying specific public health strategies [bib_ref] FilaBavi, a demographic surveillance site, an epidemiological field laboratory in Vietnam, Chuc [/bib_ref].
The overall aim of an HDSS involves developing an epidemiological surveillance site to produce basic population-based health data; serving as a background and sampling frame for specific studies, especially longitudinal studies; creating formal training capabilities, particularly for epidemiological training of research students; and providing evidence to policy-makers to support better policies and healthcare interventions [bib_ref] FilaBavi, a demographic surveillance site, an epidemiological field laboratory in Vietnam, Chuc [/bib_ref] [bib_ref] Health and demographic surveillance in rural western Kenya: a platform for evaluating..., Adazu [/bib_ref].
Nepal currently lacks the capacity to provide reliable and accurate data collection on a longitudinal basis. Therefore, this study aimed to establish an HDSS in Bhaktapur district as a collaborative effort between the Nordic School of Public Health NHV, Sweden; Kathmandu Medical College (KMC) and Nepal Medical College (NMC), Nepal. The HDSS concept in Nepal is still in the initial stage of development therefore we encountered many challenges related to practical issues around supplying imperative information for health planning and interventions. Here, we describe the preliminary experiences and findings of the newly established HDSS in Jhaukhel-Duwakot, a village area of Nepal.
# Methods
## Study site and population
Duwakot and Jhaukhel villages lie in the mid-hills of Bhaktapur district, 13 kilometers outside the capital city, Kathmandu. These villages were chosen for HDSS primarily due to their proximity to the community hospitals run by KMC and NMC and secondarily due to them being prototypical urbanizing villages by the larger towns in Nepal.
Duwakot is located 1,367 meters above sea level and has an area of 6.42 square kilometers [fig_ref] Figure 1: Map of Nepal showing Bhaktapur district [/fig_ref]. KMC operates a 70-bed community hospital in this village. Adjacent to the east of Duwakot, Jhaukhel is 1,401 meters above sea level and covers an area of 5.41 square kilometers. NMC operates a 25-bed community hospital in this village. The national government maintains a health post in each of the two villages.
Duwakot and Jhaukhel share a common geography, ethnicity and culture, and concrete roads provide both villages with accessibility to the Bhaktapur-Kathmandu Highway. Most households in Duwakot and Jhaukhel have piped water, electricity and modern communication facilities. Although both villages maintain primary and secondary schools, they lack high schools or colleges other than the medical colleges.
## Manpower recruitment
A core local management committee, consisting of an HDSS coordinator and PhD students, was formed to oversee HDSS activities. Every village in Nepal is divided administratively into nine wards. We recruited 18 female enumerators (1 per ward) for data collection all enumerators lived in the same ward where they would collect data. We recruited female enumerators mainly for better compliance and cooperation as the respondents are culturally more receptive to them. The minimum qualification required to be enumerator was completion of grade 10. We also hired two male field supervisors, one for each village.
## Household listing and social mapping
In August 2010, the surveyors were trained to (i) properly list and enumerate the households in each ward and (ii) prepare a manually drawn social map for each village. The surveyors then pretested the questionnaire in the nearby houses, emphasizing informed consent; each respondent was free to participate or end the interview at any time. Finally, trained data entry operators created a database of the list, using Epidata 3.1 software.
## Tools
The baseline census questionnaire was developed using the FilaBavi and DodaLab HDSS model that was developed in Viet Nam. The experience of management committee members in the Community Diagnosis (CD) Programmes of the medical colleges helped tailor the questionnaire to the local Nepali context. The questionnaire contained semi-structured questions on demographic parameters, such as birth, death, marriage and migration; health and health-seeking behaviours; and social, environmental and economic factors. Each question was coded for data entry. After completing the household listing, the enumerators were trained in baseline data collection, which was conducted between October and December 2010.
The socioeconomic class was defined by Kuppuswamy's socioeconomic status scale modified to the Nepalese context [bib_ref] Modification of Kuppuswamys socioeconomic status scale in context to Nepal, Ghosh [/bib_ref]. The scale takes into account the education status, occupation and family income per month to categorize the family into high, middle and low socio economic status. The income classification of the original scale in Indian currency was here converted into Nepalese Rupees by multiplying with 1.6 (1 Rs Indian = 1.60 Rs. Nepali) and also using the 2010 consumer price index of Nepal. The Reliability Coefficient (α) was 0.503 in the current study.
Illness was recorded for 4 weeks immediately preceeding the survey. Attrition of death was done on the basis of the response provided by the respondent. Multiple responses were recorded if more than one cause was provided for both morbidity and mortality.
## Monitoring, supervision, and quality control
Field supervisors, the HDSS coordinator and the PhD students supervised the collection process on a regular basis, and supervisors oversaw the interview process. To doublecheck the quality of data collection, the supervisors also conducted random, repeat interviews in 5% of the households. To rectify problems encountered in the field during data collection and to obtain maximum response and reliable data from the community, enumerators, supervisors and PhD students held regular discussions.
## Data management and analysis
We outsourced both data entry of the household listings and baseline data to a team of public health graduates, who entered it into Epidata software, version 3.1. We checked the data entry process regularly, seeking feedback and discussion about the many unforeseen problems as they arose (see below). Data analysis was conducted using Microsoft Excel 2007; SPSS Statistics 17.0; and STATA 10. We used both descriptive (percentage, mean, standard deviation) and inferential statistics (logistic regression) for data analysis. Besides these, we also computed different types of crude and specific rates and ratios.
# Ethical issues
We obtained informed verbal consent from all respondents. Ethical approval was obtained from Nepal Health Research Council (NHRC). We also briefed local administrative authorities, health personnel and political leaders about the study's objectives, and we obtained their verbal permission to conduct the survey. To ensure confidentiality, all data were secured in the HDSS office. The participating Nepalese medical institutes provided subsidized medical services to respondents who required care during the survey.
# Results
In 2010, the Jhaukhel-Duwakot Health Demographic Surveillance Site (JD-HDSS) was established in Nepal with its main office in the NMC community hospital in Jhaukhel [fig_ref] Figure 2: Photos [/fig_ref]. [fig_ref] Table 1: Household size, population size and sex ratio of JD-HDSS, Bhaktapur, 2010 [/fig_ref]. The study population covers nearly 5% of the total population of Bhaktapur district . The total population of the study area has increased nearly by 3% (annual growth rate = 0.26%) and total household number has increased by 15% after the national census 2001. The average household size of JD-HDSS is similar to the data reported by the Nepal Health Demographic Survey 2006. Households in both villages showed a 96% response rate during data collection. With few exceptions, all forms were filled in completely.
## Findings of the baseline census 2010
## Socio-demographic findings
According to the age-sex pyramids of the two villages, a majority of the population was between 10 and 30 years of age, and the birth rate was low [fig_ref] Figure 3 and: Tables 1, 2, 3present the findings, separately and combined, of the 2010... [/fig_ref] and 3B). The proportion of population is decreasing with age group after 20-24 years. It indicates that the proportion of deaths may rise in the future as a consequence of low birth rate. The median age of the population is 27 years for both males and females.
The predominant castes were Newars (36.5%), Chhetris (30.4%) and Brahmins (23.4%); and nearly 97% of the population was Hindu. Among employable individuals, 25% were students aged between 10 and 30 years, fewer than 11% worked in agriculture, nearly 20% were service holders, and 2% were unemployed. Almost one fifth (18.2%) of the individuals ≥ 6 years of age were illiterate. More than two thirds of the population was economically active and based on education, occupation and income levels attained by heads of the households, about 60% belonged to the upper-lower class (figure 4); 93% owned their own home. Around 50% of households used piped drinking water, almost all (99.5%) had electricity, and 3% lacked indoor toilet facilities.
# Fertility-related findings
Fertility-related indicators for JD-HDSS offered a good picture of overall fertility rate, with a crude birth rate of 9.7/1,000 total population and a home delivery rate of 11% [fig_ref] Table 2: Vital statistics, JD-HDSS, Bhaktapur, 2010Infant mortality rate and maternal mortality ratio are... [/fig_ref]. Mean age at marriage (± standard deviation) was 22.2 (±4.6) and 18.4 (±3.6) years for boys and girls respectively with difference in mean age at marriage of 3.4 which is similar to the national figures for rural areas [bib_ref] Nuptiality trends and differentials in Nepal, Chaudhury [/bib_ref]. Females produced offspring within 2 years of marriage (20.4 ± 3.2 years).
## Migration
Migration data shows that about 2% of the population had migrated to the area [fig_ref] Table 2: Vital statistics, JD-HDSS, Bhaktapur, 2010Infant mortality rate and maternal mortality ratio are... [/fig_ref] , mostly as family units, and the majority migrated from adjacent districts, including Dolakha (14.8%), Sindhupalchock (14.5%), Ramechap (12.5%), Kavrepalanchok (6.6%) and Kathmandu (4.9%). On the other hand, out-migration (i.e., predominantly one member per house, who moved for work or study) was 1.36% [fig_ref] Table 2: Vital statistics, JD-HDSS, Bhaktapur, 2010Infant mortality rate and maternal mortality ratio are... [/fig_ref]. Among the out-migrant population, about one fifth migrated internationally.
## Mortality
Our survey recorded no deaths among infants or children younger than 5 years of age. One third of all deaths occurred below the average life expectancy (i.e., 64 years). The crude death rate was 3.9 per 1,000 population per year. Non-communicable diseases (NCDs), including hypertension, diabetes mellitus and cancer, were major causes of death [fig_ref] Table 3: Common causes of mortality, JD-HDSS, Bhaktapur, 2010 [/fig_ref]. Based on 2001 census, among total deaths, 2.6%, 1%, 0.6% and 3.6% of deaths were accountable to heart diseases, hypertension, diabetes, and cancer respectively.
## Health and health behavior
Nearly 15% of individuals in the study area smoked cigarettes (1 in 5 among the males and 1 in 10 among the females), and about 67% of smokers belonged to an upper-lower class family (data not shown). One in 10 people reported being ill during the four weeks immediately preceding the survey [fig_ref] Table 2: Vital statistics, JD-HDSS, Bhaktapur, 2010Infant mortality rate and maternal mortality ratio are... [/fig_ref]. [fig_ref] Table 4: Top ten morbidity reported in JD-HDSS, Bhaktapur, 2010Data is for illnesses within... [/fig_ref] shows the top 10 causes of morbidity. The most common cause of illness was respiratory problems, followed by heart disease, hypertension and gastric ailments. Age-adjusted multivariate analysis of the composite prevalence of the main four NCDs (i.e., heart disease, hypertension, cancer and diabetes) shows that NCDs occur more frequently in females, Tibeto-Burman ethnic groups, agricultural workers or laborers, the illiterate and smokers [fig_ref] Table 5: Age-adjusted multivariate analysis for noncommunicable diseases [/fig_ref]. Nearly 25% of ill individuals visited traditional healers. For modern medicine, they visited the district hospital or bought medicine directly from a medicine shop; they visited local governmental outlets (e.g., health posts and the private hospitals/clinics) less frequently [fig_ref] Figure 5: Health service utilization by people during illness, Baseline Survey 2010 [/fig_ref].
## Challenges at jd-hdss
HDSS provide methods for collecting unique and continuous demographic data that describes an entire population; hence, producing reliable data is both imperative and a major challenge. Our team faced many administrative, political, geographical and social challenges during establishment of the JD-HDSS.
Before performing the baseline survey, our team conducted several rounds of discussion with concerned authorities (i.e., medical schools, governmental bodies, local authorities and political leaders) about the importance of JD-HDSS. One challenge involved justifying the need for the study and then obtaining the commitment of the authorities, which was crucial to the long-term continuation of the project.
Another critical element in establishing a well-defined surveillance site involves hiring appropriate human resources from within the study locality. The norm developed in JD-HDSS involves hiring local data enumerators and supervisors, thus establishing an easy rapport between the study population and the researchers, enhancing convenient data collection and benefiting economic activities. Although the minimum qualification was grade 10 for data enumerators and an Intermediate passed for field supervisors, bachelor or master-level candidates applied for both posts. Thus, high competition and overqualified candidates increased the challenge of selecting suitable manpower. Moreover, political leaders and bureaucrats exerted additional pressure during the recruitment process.
As JD-HDSS is located in mid-hills, participant households were scattered even within wards and they are not easily accessible from the single concrete road of the village. Indeed, access to the entire area is limited by the road facility and the poor availability of public transportation. It requires more than an hour to travel from one location to another. Moreover, Nepal lacks a systematic numbering system for its households, making it very difficult for enumerators and field supervisors to identify the exact location of some houses and, thus, hampering fieldwork.
Household members were not always available during the survey visits, necessitating a second visit. In addition, even when respondents were at home, they were often busy and unable to devote the time required to complete the lengthy questionnaire (9 different parts in 16 pages). Similarly, the questionnaire included the collection of very difficult information regarding migration. Despite these difficulties, our supervisors and enumerators were able to collect information from 96% households.
Data management is a crucial component of reliability and validation. At JD-HDSS, the preparation of data for analysis included several stages. First, completed forms were kept in the JD-HDSS office, where staff members checked that they were filled out completely. Next, the completed forms were sent to the data entry operators. If they found an error, the data entry operators sent the forms back to the field for correction. For example, some forms were completed in mixed language (i.e., both English and Nepali). Frequent cross-checking between computer entries and Data is for deaths that had occurred in the preceeding year before the survey. Attrition of death was done on the basis of the response provided by the respondent. Multiple responses were recorded if more than one cause was provided. Respiratory diseases included both chronic obstructive lung diseases and pulmonary infections.\ filled forms ensured accuracy and completeness. This type of scrutiny increased the focus and awareness of the data entry operators. Similarly, all concerned staff members received guidance on the ethical issues regarding the storage and use of data. All clean data sets were shared among the concerned authorities of JD-HDSS in a format that was user-friendly for scientific publication. Because keeping completed forms and other necessary documents in safe, long-term storage posed another challenge, it was necessary to develop a code of conduct that governed access and sharing of the data.
# Ethical issues
We encountered many ethical issues during the baseline survey. Although such issues did not reach the sensitivity level of clinical or drug trials, respecting the dignity, feelings, freedom and confidentiality of all respondents was essential. While establishing an HDSS in a selected area, one hurdle involves the fact that many other groups may already have visited the households to collect data for different purposes. Consequently, study participants may become irritated by the attention they receive for monitoring purposes and they may demand immediate direct benefits. Because team members were unable to provide medication to ill participants, such individuals wanted assurance about the long-term benefits of participating in the survey. Team members explained the plan to develop their village as a health model village in Nepal, and households received 25%-50% discounts on hospital services at Kathmandu Medical College and Nepal Medical College.
We also encountered many issues regarding migration. Some participants did not wish to disclose the reasons for their migration (e.g., political pressure, family issues). Similarly, they did not want to share information about monthly income, some diseases (e.g., uterine prolapse), sexually transmitted infections and HIV/AIDS.
Although respondents received no in-hand benefits for participating in the baseline survey, the HDSS team is now considering awarding each household with in-hand benefits (e.g., soap, toothpaste, toothbrushes) during data collection by enumerators and posters, pamphlets and health education when they complete the baseline study.
In the long-term, it simplifies the collection of data and also ensures that the project will remain sustainable.
# Discussion
## Relevance of jd-hdss
This HDSS in progress is the first in Nepal with focus on health issues. A Household Registration System with HDSS methodology had been in the running in the late 1990's in the Southern Nepalese town of Chitwan in collaboration with Population Studies Centre. However, that project mainly investigated migration issues and followed even those who had out-migrated from the study area after the start of the project. The intention of this paper is to share the methodology and initial experience of establishment of a HDSS in Nepal and we aim to present longitudinal data after completion of planned subsequent rounds. In terms of the population size, compared to other HDSSs such as Agincourt in South Africa and Matlab in Bangladesh, JD-HDSS is a modest endeavor in terms of the area covered and population size [bib_ref] Research into health, population and social transitions in rural South Africa: Data..., Kahn [/bib_ref]. Nonetheless, as put forward by Byass and colleagues, there is no exact formula or statistical ways to determine the optimal sample size of a HDSS. Similarly, the external validity of a study is also an important parameter and in that sense, JD-HDSS is a prototype of the villages near the cities in Nepal which are fast growing into peri-urban or suburban stature. However, given the ethnic and geographic diversities of Nepal, it cannot claim to be representative of all the ethnic and geological facets of the country. Nevertheless, from the point of view of study objectives, the selection of HDSS area is arguably well suited because historically establishment of a HDSS has always been tailored to the local needs. The HDSS in Western Kenya, for example, was initiated as a field study site for testing the effectiveness of insecticide-treated nets [bib_ref] Health and demographic surveillance in rural western Kenya: a platform for evaluating..., Adazu [/bib_ref].
In that regard, JD-HDSS is optimal in terms of its current focus on urbanization and NCD risk factors, as the chosen villages are rapidly undergoing sociodevelopmental transitions. And, as shown by our baseline survey from the HDSS, in its early stage of demographic transition, Nepal faces a double burden of diseases. This is characterized by simultaneous infectious disease and an increasing NCD burden due to urbanization and changing lifestyles. Without preventive measures to combat this trend, Nepal will experience a significantly increased NCD burden. Due to current emphasis on curative care rather than preventive measures, Nepal does not identify the root causes of disease (i.e., hidden social, cultural and demographic aspects). Different cross-sectional studies conducted at various intervals have mentioned these risk factors but failed to establish casual relationships between risk factors and disease [bib_ref] Obesity prevalence in nepal: public health challenges in a low-income nation during..., Vaidya [/bib_ref]. Similarly, Nepal has promulgated laws for registration of vital statistics (i.e., birth, death, marriage, divorce and migration) but has not established the necessary reporting mechanisms yet. Consequently, Nepal needs to establish HDSS to monitor different demographic events: birth; deaths; marriage; migration and health events: types of sickness; hospital visits etc. within a defined geographical community, and also provide a podium for interventional studies regarding subpopulations [bib_ref] Health and demographic surveillance in rural western Kenya: a platform for evaluating..., Adazu [/bib_ref]. Nepal has experienced rapid urbanization (i.e., nearly 4.7% per year). Located near Kathmandu, the capital city, JD-HDSS has already begun the process of shifting from a rural to urban community. Changing lifestyles, from traditional to modern, significantly impact demographic and social structures. Additionally, in-and outmigration impacts JD-HDSS, whose positive net migration rate (0.0085%) indicates population gain. The current baseline survey determined that NCDs are also prevalent alongside communicable diseases, noting that the population, not unexpectedly, has a double burden of disease. Thus, the JD-HDSS study site provides a good platform for studying disease burden from the dual aspects of both health promotion and disease prevention.
## Findings from the jd-hdss baseline study
The crude birth and death rates of the JD-HDSS are 35% and 53% lower, respectively, compared to national figures (CBR =27.7 per 1,000 population/CDR =9.7 per 1,000 population) estimated in the year 2008. Total age dependency ratio (younger than 15 years and older than 60 years) was 31 per 100 working-age population (15-60 years), of which 24 per 100 belonged to under 15 years of age. Similarly, the baseline survey observed no mortality in infants, children younger than five years of age, and mothers. According to the Nepal Health Demographic Survey 2011, the national infant and under-five mortality rates are 46 and 54 per 1000 live births, but a wide variation has been noted, for example, according to place of residence (urban and rural rates are 38 and 55, 45 and 64 respectively). Thus the reason for not finding any child or maternal death could be because of improved living standard in the selected study area, easy access to healthcare services (including access to two community hospitals and a health post), the low illiteracy rate, increased income (only 2.4% lower class), and healthier nutrition. It could as well be because of smaller study population.
The study also determined that more than 90% of children were born in health institutions, more than three times the rate disclosed in the preliminary report of the Nepal Health Demographic Survey 2011, where only 58% of mothers received prenatal care from a doctor or nurse and only 28% of all births occurred in health facilities. Although JD-HDSS has met the Millennium Development Goal (MDG) target (i.e., 60% of all births attended by a skilled provider), Nepal itself is far from attaining that goal. About 33% of deaths occurred in individuals younger than 65 years of age in the study area, about two times less than that reported by the 2001 national census. The census also reported higher life expectancy in Bhaktapur district (71.33 years) compared to the Kathmandu and Lalitpur districts (69.5 years and 67.10 years, respectively) and the entire country (64.1 years) .
The net migration rate for JD-HDSS was 0.0085% (inmigration = 2.25; out-migration = 1.36). In-migration increased from 13.4% to 26.8% between 1971 and 2001, respectively. Rural-to-urban migration predominates in Nepal, especially in the large cities of Kathmandu, Lalitpur and Bhaktapur districts. According to national census 2001, those districts received 71.8%, 82.7% and 44.6% in-migrants, respectively [bib_ref] Internal migration in Nepal: Population Monograph of Nepal, Bk [/bib_ref] [bib_ref] Bangkok: The Economic and Social Commission for Asia and the Pacific, Bk [/bib_ref]. The same census showed, the five major reasons for in-migration -business, agriculture, service, education and marriage-mirrored our findings (data not shown) [bib_ref] Internal migration in Nepal: Population Monograph of Nepal, Bk [/bib_ref] [bib_ref] Bangkok: The Economic and Social Commission for Asia and the Pacific, Bk [/bib_ref]. In our study, the majority (91%) of out-migrated people explained that they migrated to increase earning power and get a better education.
The annual health report for fiscal year 2009-2010 listed gastritis, respiratory problems, and injuries among Nepal's 10 leading morbidities. About 53.22% of the total population visited the outpatient department in Bhaktapur district, of which 57.27% were male. Among them, 9.22% sought outpatient treatment for skin diseases; 6.17% for gastritis; 3.76% for cardiovascular diseases; 3.18% for dental problems, including pain; 3.70% for headache; 2.57% for hypertension and 1.91% for pain. JD-HDSS observed similar morbidity patterns.
In the JD-HDSS study area, NCD prevalence among individuals older than 30 years was 4.32% associated with several factors, including sex, ethnic group, occupation and education. Currently, few community-based studies have investigated NCD risk factors in Nepal. One study, conducted in Eastern Nepal, reported an NCD prevalence rate of 6% in adult males, and a 2008 survey studying NCD reported that more than 80% of respondents exhibit one or more risk factors [bib_ref] Prevalence of coronary heart disease in the urban adult males of eastern..., Vaidya [/bib_ref]. A recent hospital-based study, conducted in 31 health institutions in Nepal, reported that 36.5% of patients suffer from NCD and share of female was 52.4% of the total patients. Our study showed that smoking carries a 26% risk for developing NCD (unadjusted odds ratio1.26 [95% CI: 0.97-1.63]) compared to 60% reported by a recent hospital-based study in Nepal. Similar to our findings, that study also reported that the adjusted odds ratio for smoking was not statistically significant.
Ongoing challenges for NCD control in Nepal include political instability, poor health literacy, greater focus on curative aspects and rapid urbanization [bib_ref] Tackling cardiovascular health and disease in Nepal: epidemiology, strategies and implementation, Vaidya [/bib_ref]. The control approach works well if lifestyle factors are modified through effective health education intervention and tobacco control policies. To forecast cumulative risk, future studies should investigate risk factor clusters (i.e., a combination of two or more risk factors) for NCD development rather than focus on single-factor risks [bib_ref] Clustering of chronic noncommunicable disease risk factors among selected Asian populations: levels..., Ahmed [/bib_ref].
# Limitations
Although our success in establishing Nepal's first HDSS provides a reliable sampling framework for subordinate studies and builds mutual trust between village residents and researchers, the baseline survey includes several limitations that require further attention. Because we obtained our baseline data from any family member who was older than 18 years of age, our data may be both over-and under-reported. There is also a possibility of bias regarding both recall and selection. Selection bias may arise due to the selection of convenient respondents from whom the enumerators can obtain information easily, whereas supervision bias may result from time constraints regarding form checking. Because the JD-HDSS geographic area was selected purposively, our survey covered only two village developmental committees; hence, the findings should be generalized cautiously to populations beyond the surveyed population [bib_ref] Using the INDEPTH HDSS to build capacity for chronic non-communicable disease risk..., Ng [/bib_ref].
To elicit information on death and its cause, the survey interviewer asked the question "Has any member of your family died within the year?" If the answer was yes, respondents were asked about age, sex and the cause of death. So there is possibility of measurement bias, e.g., data enumerators might fail to record all deaths that lead to under reporting of deaths and hence the possibility why the crude death rate in the JD-HDSS is below the national [fig_ref] Figure 3 and: Tables 1, 2, 3present the findings, separately and combined, of the 2010... [/fig_ref]. Also, the emotional attachment with the death of a family member may make the respondents not wanting to talk about it. Furthermore, the enumerators do not ask for any details regarding deaths. In addition, there is the possibility of recall bias, i.e., the respondents cannot remember exact date of deaths.
We also found that only 7 of 10 deaths were reported to the village office. It indicates that the vital registration system does not cover all deaths and fails to provide accurate data. This underlines the importance and need of an HDSS in Nepal, which enables recording of deaths on a regular basis. Admittedly, the verbal autopsy method would have been a better tool to elicit the causes of deaths. We were unable to use verbal autopsy for the baseline study because of lack of trained manpower but do intend to use them in future surveys.
Additional interviewer bias can also come from the enumerators as many of them were overqualified for enumeration work and they can add their own thoughts in to a response while recording it. Similarly, the response to the enumerators belonging to a different political ideology can hamper the respondents' answer during the interview. Additionally, the length of the questionnaire may have contributed hurried and often untrue or incomplete responses. Similarly, lack of 'on-the-spot' benefit could have biased the respondent's reply.
Finally, provision of ancillary care is an ethical dilemma for which there are no clear-cut guidelines or solutions [bib_ref] Demographic and health surveillance: longitudinal ethical considerations, Carrel [/bib_ref]. Over-inflation of the HDSS budget is an important possibility to consider while providing such care.
## Strengthening of the jd-hdss
We have used basic technology for village mapping and data collection in the baseline census. Mapping can be done more scientifically in the future with the use of GPS technology like in FilaBavi and many African HDSSs [bib_ref] FilaBavi, a demographic surveillance site, an epidemiological field laboratory in Vietnam, Chuc [/bib_ref] [bib_ref] Health and demographic surveillance in rural western Kenya: a platform for evaluating..., Adazu [/bib_ref]. This could also have paved way for mapping of the environment coverage of the HDSS [bib_ref] Mapping the environmental coverage of the INDEPTH demographic surveillance system network in..., Tatem [/bib_ref]. Cardiff Teleforms, used for example in Western Kenya, could also be helpful if there is possibility of technical support [bib_ref] Health and demographic surveillance in rural western Kenya: a platform for evaluating..., Adazu [/bib_ref]. Even data entry and analysis software can be upgraded to maintain and produce quality data [bib_ref] Research into health, population and social transitions in rural South Africa: Data..., Kahn [/bib_ref]. Effort should also be made towards improving the internal validity of our data collection technique by using more widely accepted methods such as Verbal Autopsy for information on cause of mortality [bib_ref] Research into health, population and social transitions in rural South Africa: Data..., Kahn [/bib_ref] [bib_ref] Using health and demographic surveillance to understand the burden of disease in..., Pronyk [/bib_ref]. Expansion of the study area itself to include a more urban area can yield interesting comparisons in demographic and health parameters. Finally, effort should be made to eventually make this HDSS an IN-DEPTH member.
## Future studies
A new round of census survey will begin in September 2012, followed by another survey every six months. JD-HDSS is also currently conducting two longitudinal subordinate studies, one related to tobacco smoking behavior among 500 adolescents and the other related to knowledge, attitudes and practice of cardiovascular disease in people aged 25-59 years. Additional subordinate studies on maternal health and nutritional status are in the pipeline.
In agreement with the thoughts put forward by Baiden, the JD-HDSS can be a good field site for other scholarly interests as the project is primarily of academic nature [bib_ref] Demographic surveillance sites and emerging challenges in international health, Baiden [/bib_ref]. Presence of gaps in socio-economic parameters and diversity in health-care utilization, studies on issues such as socio-economic determinants of health, health equity and health system reform are interesting tentative projects in the JD-HDSS [bib_ref] Socio-economic status and health inequalities in rural Tanzania: evidence from the Rufiji..., Mwageni [/bib_ref] [bib_ref] Health system reform and the role of field sites based upon demographic..., Tollman [/bib_ref]. As done in the Agincourt HDSS, triangulation of our data with the relevant national data could also be a potentially useful exercise particularly in the context of urbanization and its complexities.
# Conclusions
In tandem with the current baseline survey, the study reported here demonstrates that it is possible to establish an HDSS in a village area of Nepal to collect accurate and reliable data. Such prospective data on health and demography also provides a platform for public health research and the evaluation of community-based health policies and practices. Results from the baseline survey show that both maternal and child health in the surveillance site exceed that of the entire country. The baseline survey also reported on risk factors associated with NCDs. Thus, studying NCD burden in the HDSS can yield reliable epidemiological data for prevention and control through longitudinal studies.
[fig] Figure 3 and: Tables 1, 2, 3present the findings, separately and combined, of the 2010 baseline census. JD-HDSS includes 2,712 households containing a total of 13,669 individuals ( [/fig]
[fig] Figure 1: Map of Nepal showing Bhaktapur district (insert) and the location of the Health Demographic Surveillance Site (HDSS) inDuwakot and Jhaukhel villages in Bhaktapur district (right)[11]. [/fig]
[fig] Figure 2: Photos (from top left to lower right): HDSS office (Nepal Medical College, Jhaukhel) and the office room; training session; enumerators during training; the making of social maps; enumerator during an interview (baseline survey training and action). [/fig]
[fig] Figure 3: Population pyramid of the Duwakot (A) and Jhaukhel (B) development committees, Baseline Survey 2010. [/fig]
[fig] Figure 4: Socio-economic classification of the households in the Health Demographic Surveillance site at Jhaukhel and Duwakot (JD-HDSS). The socioeconomic class was defined by Kuppuswamy's socioeconomic status scale modified to the Nepalese context[13]. The scale takes into account the education status, occupation and family income per month to categorize the family into high, middle and low socio economic status. The income classification of the original scale in Indian currency was here converted into Nepalese Rupees by multiplying with 1.6 (1 Rs Indian = 1.60 Rs. Nepali) and also using the 2010 consumer price index of Nepal. The Reliability Coefficient (α) was 0.503 in the current study. [/fig]
[fig] Figure 5: Health service utilization by people during illness, Baseline Survey 2010 (multiple answers). [/fig]
[table] Table 1: Household size, population size and sex ratio of JD-HDSS, Bhaktapur, 2010 [/table]
[table] Table 2: Vital statistics, JD-HDSS, Bhaktapur, 2010Infant mortality rate and maternal mortality ratio are not shown as there were no deaths reported. Illness was recorded for 4 weeks immediately preceeding the survey. Out-migration did not cover the families in which all the family members of the household had migrated. [/table]
[table] Table 3: Common causes of mortality, JD-HDSS, Bhaktapur, 2010 [/table]
[table] Table 4: Top ten morbidity reported in JD-HDSS, Bhaktapur, 2010Data is for illnesses within four weeks prior to the survey. Multiple responses were recorded if more than one cause was provided. Respiratory diseases included both chronic obstructive lung diseases and pulmonary infections. Non-communicable diseases such as hypertension and diabetes were selfreported and are prevalent alongside communicable diseases. [/table]
[table] Table 5: Age-adjusted multivariate analysis for noncommunicable diseases [/table]
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Why monitor the neonatal brain—that is the important question
Published online: 1 April 2022✉email:[email protected] online: 1 April 202210.1038/s41390-022-02040-9Received: 1 March 2022 Accepted: 6 March 2022COMMENT OPEN Why monitor the neonatal brain-that is the important question
A key goal of neonatal neurocritical care is improved outcomes, and brain monitoring plays an essential role. The recent NEST trial 1 reported no outcome benefits using aEEG monitoring compared to clinical seizure identification among neonates treated for seizures. However, the study failed to prove the effects of monitoring on seizure treatment in the first place.
Neonates with acute neurological disorders, such as encephalopathy due to hypoxia-ischemia, will commonly undergo continuous electroencephalography (EEG) monitoring to assess brain recovery and detection of seizures. EEG monitoring is necessary for a reliable diagnosis and monitoring of neonatal seizures 2 , because the majority of EEG-confirmed neonatal seizures have no clinical signs, while EEG monitoring may confirm that many clinical events are non-epileptic in origin [bib_ref] Interobserver agreement in neonatal seizure identification, Malone [/bib_ref]. The International Neonatal Consortium, European Medicines Agency, US Food and Drug Administration, Brighton Collaboration, the International League Against Epilepsy, and American Clinical Neurophysiology Society agree that all studies involving the treatment of neonatal seizures should evaluate seizures from the EEG recordings 2,4,5 .
Before these international recommendations, it was common for clinicians to question the added value of (a)EEG monitoring in newborn care. The NEST trial 1 attempted to address this question by assessing whether neurodevelopmental outcomes are improved by treating seizures with the aid of amplitudeintegrated EEG monitoring (aEEG) compared to treating seizures identified by clinical recognition only. The underlying assumption was that seizure identification and management reduces secondary brain injury and improves neurodevelopment , and that aEEG facilitates seizure diagnosis and treatment. The study found no evidence of improved neurodevelopmental outcomes, and secondary analyses showed reduced cognitive outcomes in the group with aEEG monitoring. The secondary outcome findings can be linked to many well-established confounders, only some of which were mentioned in the paper. However, the most surprising finding of this study is the lack of difference in overall seizure burden between groups treated with aEEG monitoring support versus those with clinical consideration only.
It is now widely recognized that (a)EEG monitoring and seizure treatment should commence as early as possible because the seizure burden peaks during the first 24 h in infants with hypoxicischemic encephalopathy (the primary cause of neonatal seizures) with subsequent gradual resolution [bib_ref] Neonatal seizure management: is the timing of treatment critical?, Pavel [/bib_ref] [bib_ref] Characterisation of neonatal seizures and their treatment using continuous EEG monitoring: a..., Rennie [/bib_ref]. This spontaneous resolution of seizures over time means that a late start of treatment would require prohibitively large study cohorts and only show a treatment response with limited clinical significance 8 . In the NEST study, aEEG was initiated after 24 h; therefore, NEST likely missed the bulk of the seizure burden, and it only assessed the remains of the spontaneously decaying seizures. Thus, the NEST study design per se precluded an opportunity to see an actual treatment effect in either study arm [bib_ref] Treatment trials for neonatal seizures: the effect of design on sample size, Stevenson [/bib_ref].
In addition to missing seizures due to the late onset of aEEG monitoring, the EEG assessment method per se used in the NEST study introduces a major confounder in seizure burden estimates . Although aEEG is often useful for clinical brain monitoring, including the recognition of longer seizures, international guidelines have established that the aEEG alone cannot provide accurate enough estimates of seizure burden. The NEST study amplifies this bias by only considering the longer seizures that were evident on the compressed aEEG trace that likely led to a considerable proportion of the seizure burden going undetected, untreated, and not accounted for in the analyses. Moreover, the report does not provide key details regarding the aEEG, for instance, which recording channels were used, how aEEG review was actually done at the bedside, whether seizure detection algorithms were (or were not) used, when treatment(s) were administered, and how seizure burden and response to treatment were subsequently quantified. Furthermore, analyses of aEEG background patterns are not reported despite their strong associations with outcome [bib_ref] Recovery of amplitude integrated electroencephalographic background patterns within 24h of perinatal asphyxia, Van Rooij [/bib_ref].
Overall, the NEST study portrays a grand challenge in all contemporary medical research. The rapidly changing clinical landscape and accumulating research data make it difficult to design new studies with influential results. This is particularly true for neonatal research where the use of long-term neurodevelopment outcomes results in very long study cycles. Consequently, the initial study question may have lost its relevance by the time the trial is completed. In the case of NEST, the concurrently accumulated evidence rendered the initial study question a classic straw man hypothesis: The study was designed to test the hypothesis that aEEG improves neurodevelopmental outcomes by improving seizure identification and seizure management, thereby reducing seizure burden . Instead, the study shows that aEEG begun >24 h after delivery did not affect treatment practices and outcomes, but it does not really address the question of whether or how early use of EEG monitoring impacts treatment success, i.e., seizure burden en route to better outcomes. Notably, this situation is changing rapidly worldwide, with further training of personnel as well as more widespread use of EEG devices with adequately validated algorithms for automated seizure detection [bib_ref] A machine-learning algorithm for neonatal seizure recognition: a multicentre, randomised, controlled trial, Pavel [/bib_ref].
Why is this issue important to raise? We believe that there is a risk that the NEST study question per se may lead clinicians to erroneously conclude that EEG monitoring is unhelpful in neonatal neurological management. This misleading effect may be worse in low-and middle-income countries where the current upskilling of neonatal units is calling for de novo implementation of brain monitoring routines. Notably, the authors of NEST study concluded that "EEG remains the reference standard for the detection of neonatal seizures, and essential in the validation of neonatal research." This important statement carries the readership onto the grand challenge question of neonatal neurological care: Why monitor the neonatal brain? Routine clinical practice has shown that EEG monitoring of neonates supports and guides individualized patient care by providing a functional measure of brain recovery after injury, reliable detection of seizures, and surveillance of sleep-wake cycling, as well as many other contextrelevant observations [bib_ref] Recovery of amplitude integrated electroencephalographic background patterns within 24h of perinatal asphyxia, Van Rooij [/bib_ref] [bib_ref] A machine-learning algorithm for neonatal seizure recognition: a multicentre, randomised, controlled trial, Pavel [/bib_ref] [bib_ref] Neurocritical care for neonates, Glass [/bib_ref]. In addition, several novel aEEG and EEG biomarkers are being developed that hold promise for making EEG a proximal outcome measure for individualized patient care, benchmarking clinical trials, as well as predicting later neurodevelopmental compromise. These benefits are irrefutable and widely acknowledged, and conceivably, trying to measure their links to neurodevelopmental outcomes without considering the possible intermediary or causal mechanisms is unproductive. Indeed, there is a widely shared bedside experience in neonatal intensive care units that trusts in a well-known mechanism, the latent Hawthorne effect [bib_ref] Neurocritical care for neonates, Glass [/bib_ref] [bib_ref] The Hawthorne effect: a randomised, controlled trial, Mccarney [/bib_ref] : mere attention to the newborn brain leads to improved newborn care. The left side flowchart summarizes rationale in the NEST study and its relationship with the international guideline of seizure detection (right side flowchart). Colors from red through orange to green code for established reliability in seizure detection. The arrows depict the assumed causal links from the clinical suspicion to the neurodevelopmental outcome. The green arrows on the left side show links with prior research evidence. The gray arrow shows the critical a priori missing evidence that should have been known before commencing the NEST study. Arrows on the right side show the links that were examined in the NEST with negative findings. Notably, NEST study relies on seizure detection by aEEG that only has modest-or low-level reliability. This effectively precludes the potential to use NEST design for examining its study question. *Diagnostic certainty according to Brighton Collaboration case definition of neonatal seizures. Level 1-gold standard, definite seizure (seizures confirmed on EEG with or without clinical manifestations); level 2-probable seizure (aEEG confirmed or clinically assessed focal clonic or focal tonic seizures); level 3-possible seizure (clinical events suggestive of epileptic seizures other than focal clonic or focal tonic seizures); level 4-not seizure (reported clinical events that do not meet case definition); level 5-not seizure (clinical events evaluated by EEG and diagnosed as not a seizure).
Pediatric Research (2023) 93:19 -21
© The Author(s) 2022
ACKNOWLEDGEMENTSFUNDINGOpen Access funding provided by University of Helsinki including Helsinki University Central Hospital.COMPETING INTERESTSS.V. is an unpaid advisor for Stratus and ANT. R.M.P. is an investigator for studies with UCB and does consultancy work for Kephala LTD, Ireland. She served as a Speaker and/or on Advisory Boards for Natus, GW, Eisai, and UCB. G.B.B. has a consultancy with UCB and Nihon Kohden to provide advice on neonatal EEG monitoring. She is a co-founder of a start-up company Kephala LTD, which provides EEG reviewing services for industry and academia. M.R.C. serves as a Speaker and/or on Advisory Boards for Eisai, UCB, and Biocodex, and does consultancy work for Sanofi Aventis. J. M.W. receives an honorarium for her role as Associate Editor for Epilepsia, advisory board for South African branch of Sanofi. S.A. receives an honorarium for his role as associate Editor for Epilepsia. He has served as consultant or received honoraria for lectures from Angelini Pharma, Biocodex, Biomarin Encoded, Eisai, GRINtherapeutics, GW Pharma, Neuraxpharma, Nutricia, Orion, UCB Pharma, Ultragenyx, Xenon, Zogenix. He has been an investigator for clinical trials for Eisai, Marinus,TakedaADDITIONAL INFORMATIONCorrespondence and requests for materials should be addressed to Sampsa Vanhatalo.Reprints and permission information is available at http://www.nature.com/ reprintsPublisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons. org/licenses/by/4.0/. |
Pioglitazone use in patients with diabetes and risk of bladder cancer: a systematic review and meta-analysis
# Introduction
Bladder cancer has become a common cancer worldwide and ranks as the ninth most frequently diagnosed cancer. In 2012, approximately 430000 new cases of bladder cancer were diagnosed. Cigarette smoking, specific exposure to arylamine, and chronic schistosoma infection are the most associated factors with increased risk for bladder cancer. In recent years, diabetes mellitus (DM) has been reported to increase the risk of bladder cancer. Meanwhile, pioglitazone, an antidiabetic agent of the thiazolidinedione (TZD) class, which is broadly used for glycemic control in patients with type 2 diabetes mellitus (T2DM), has been reported to increase the risk of bladder cancer as well. At first, the risk was reported in male rats, then, a series of clinical studies was performed to identify this relationship. However, the conclusions of published observational clinical studies remain confusing. This may result from problems associated with study design, limited sample size, and a lack of attention to potential biases. In 2016, one randomized controlled trial (RCT) (PROactive) ended its 10-year observational follow-up drawing a conclusion that the imbalance in bladder cancer cases observed during the double-blind period did not persist, and there was no overall increase in malignancies. But only one RCT result could not end the argument. In 2017, another RCT (TOSCA.IT) also reported null association between pioglitazone and bladder cancer, but the number of bladder cancer cases in this RCT was only 16, which may not be convincing. A large cohort study including 193099 people by Lewis et al also reported no statistically significant increased risk of bladder cancer associated with pioglitazone use. However, the updated US Food and Drug Administration review in 2016 concluded that use of T2DM medicine pioglitazone may be linked to an increased risk of bladder cancer, [bib_ref] Pioglitazone after ischemic stroke or transient ischemic attack, Kernan [/bib_ref] which conflicted with the newly updated RCT results by Erdmann et al, Vaccaro et al, and cohort study by In the most recently updated meta-analysis conducted by Davidson and Pan, [bib_ref] An updated meta-analysis of pioglitazone exposure and bladder cancer and comparison to..., Davidson [/bib_ref] they suggested the resurrection of the use of pioglitazone, but failed to perform sensitivity analysis and simply pooled observational studies together without excluding the duplicate population and low-quality studies [fig_ref] Table 1: Characteristics of observational studies of pioglitazone and bladder cancer Research Database [/fig_ref]. In another recent meta-analysis by Filipova et al, [bib_ref] Pioglitazone and the risk of bladder cancer: a meta-analysis, Filipova [/bib_ref] they failed to give any details about quality assessment and simply pooled RCT and observational studies together without excluding the duplicate population and low-quality studies. They did not perform subgroup analysis either, though his result showed significant heterogeneity. Other meta-analyses are not convincing mainly because inadequate evidence acquisition, poor methodology, and lack of appropriate quality assessment. Consequently, despite a number of observational studies and previous reviews, [bib_ref] An updated meta-analysis of pioglitazone exposure and bladder cancer and comparison to..., Davidson [/bib_ref] [bib_ref] Pioglitazone utilization, efficacy & safety in Indian type 2 diabetic patients: a..., Pai [/bib_ref] [bib_ref] Thiazolidinediones and associated risk of bladder cancer: a systematic review and meta-analysis, Turner [/bib_ref] [bib_ref] Pioglitazone prescription increases risk of bladder cancer in patients with type 2..., He [/bib_ref] whether pioglitazone increases the risk of bladder cancer or not still remains an enigma. We conducted a systematic review and meta-analysis of all eligible RCTs, cohort studies, and nested case-control studies about pioglitazone use in patients with diabetes and bladder cancer in order to identify a more precise and reliable relationship between pioglitazone and risk of bladder cancer. Furthermore, we also examined whether the association between them converts according to sensitivity analysis.
# Materials and methods
## Publication search
This meta-analysis was performed according to the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines. [bib_ref] Preferred reporting items for systematic reviews and meta-analyses: the PRISMA statement, Moher [/bib_ref] We carried out a search in PubMed, EMBASE, Scopus, Web of Science, Cochrane register, and Chinese National Knowledge Infrastructure databases, covering all the eligible papers published up to January 24, 2018. The search strategy included terms for outcome (bladder neoplasm* or bladder cancer* or bladder tumor* or bladder tumour*) and exposure (pioglitazone* or TZD* or thiazolidinedione*). We carefully evaluated every potentially relevant publication by examining their titles and abstracts.
All the studies matching the inclusion criteria were retrieved. The references from retrieved articles and reviews were also thoroughly checked to identify any additional relevant studies. All analyses were based on previous published studies, thus no ethical approval and patient consent are required.
## Inclusion criteria
Studies included in this meta-analysis had to meet all the following criteria: a) an RCT design or a cohort design or a nested case-control design; b) the population included in studies were patients with diabetes; c) one of the exposures of interest was pioglitazone; d) one of the outcomes of interest was incidence of bladder cancer and the result is recorded in a reliable database or using ICD codes of cancer; e) studies provided rate ratio, hazard ratio (HR) or standardized incidence ratio (SIR) with their 95% confidence intervals (CIs), or sufficient data to calculate them; and f) they were of high quality according to the Newcastle-Ottawa scale (NOS) (more than 6) 35 or were assessed to have low risk of bias, using the recommended tool in the Cochrane Handbook for Systematic Reviews of Interventions.Research on mortality rates of bladder cancer was not included because it could be unpredictably confounded by survival-related factors. We also did not consider studies in which the population included healthy people because diabetes was also considered to increase the risk of bladder cancer. If multiple publications from the same study population were available, the most recent and detailed study was entitled to be included in this meta-analysis.
## Data extraction
Two authors extracted data independently by using a predefined data collection form, with disagreements being resolved by consensus. For each study, the following characteristics were collected: first author's name, year of publication, the country in which the study was carried out, participant characteristics (age and sex), study design, study population, range for follow-up, number of events and non-event subjects exposed to pioglitazone and comparison, medication use in comparison group, estimate effects with their 95% CIs and covariates adjusted for in the analysis. From each of the studies, we optionally excerpted the relative risk estimate that was adjusted for the greatest number of potential confounders. In dose effect analysis, we defined low dose as ≤8268 mg or ≤10500 mg or ≤14000 mg; moderate dose as 10501-28000 mg or 14001-40000 mg; high dose as >28000 mg or >40000 mg.
Quality assessment of searched papers was also undertaken independently by two authors according to the NOS for
## 1629
Pioglitazone use in patients with diabetes and risk of bladder cancer observational studies, and Cochrane Tool Review Manager 5.3 for RCTs. The disagreements between authors were resolved by discussion and consensus.
# Statistical methods
Studies that reported various measures of RR were included in this meta-analysis: rate ratio, HR, and SIR. In practice, these three measures of effect yield similar estimates of RR because the absolute risk of bladder cancer in patients with diabetes is low.
Overall RR estimates with their corresponding 95% CIs were calculated with the DerSimonian and Laird 37 random effects models, which consider both within-study and between-study variation. Heterogeneity among studies was measured by Q test and quantified by I 2 with higher value indicating a greater degree of heterogeneity. Random effects model was chosen to analyze the data. [bib_ref] On random-effects meta-analysis, Zeng [/bib_ref] Publication bias was assessed using Begg's test [bib_ref] Operating characteristics of a rank correlation test for publication bias, Begg [/bib_ref] (rank correlation method) and Egger's test (linear regression method). 40 P<0.05 was considered to be representative of a significant statistical publication bias. Dose effect analysis of pioglitazone was conducted if data were sufficient. Sensitivity analyses were also performed to assess the stability of obtained results with a single study deleted each time to manifest the influence of the individual dataset to the pooled RR. All of the statistical analyses were performed with STATA 14.0 (StataCorp LP, College Station, TX, USA), using two-sided P-values. [fig_ref] Figure 1: Flowchart of study assessment and selection [/fig_ref] outlines our study selection process. Briefly, after removing duplicates, the search strategy generated 561 articles. Of these, the majority was excluded after the first screening based on abstracts or titles, mainly because they were reviews, basic research, case-control studies, or obviously not relevant to our analysis. After full-text review of 21 papers, 14 studies were excluded for the following reasons: low NOS quality score; [bib_ref] Association between pioglitazone and bladder cancer among patients with type II diabetes:..., Hsu [/bib_ref] [bib_ref] Assessing the association of pioglitazone use and bladder cancer through drug adverse..., Piccinni [/bib_ref] outcomes were not bladder cancer; 43,44 duplicate population; used rosiglitazone as reference group; 45 used non-DM people as reference group; 46,47 without reference; 11 obviously abnormal data (details of exclusion criteria in [fig_ref] Table 1: Characteristics of observational studies of pioglitazone and bladder cancer Research Database [/fig_ref]. We also identified five publications through checking reference lists of retrieved studies and reviews. Thus, a total of 12 cohort studies or nested case-control studies, 7-10,12-15,19,23-25 which met the inclusion criteria, were included in the meta-analysis. Two RCT studies were also identified in this process and were systematically reviewed.
# Results
## Literature search
Publications identified through database searching (n=674)
Publications after duplicates removed (n=561)
Full text identified and screened for potentially relevant publications (n=21)
Publications identified through checking reference list of retrieved studies and reviews (n=5)
Publications included in this meta-analysis (n=12)
Publications excluded after screening titles and/or abstracts generally because they were reviews, basic researches or obviously irrelevant to our study (n=540)
Publications excluded because: low NOS quality score (n=5) Outcomes were not bladder cancer (n=2)
Use rosiglitazone or non-DM patient as reference group (n=3) Duplicate population (n=2) Without reference group (n=1) Obviously abnormal data (n=1) Two RCT studies, PROactive and TOSCA.IT, which reported bladder cancer cases in patients with diabetes, were eligible for this study. As it is meaningless to perform a metaanalysis for two studies, we systematically reviewed them. Characteristics including population, interventions, years of follow-up, and number of cases are shown in [fig_ref] Table 2: Dose effect analysis of relative risks for the association between pioglitazone and... [/fig_ref]. Risk of bias assessment of the studies is demonstrated in [fig_ref] Figure 1: Flowchart of study assessment and selection [/fig_ref].
The PROactive study was performed in patients with T2DM who were aged 35-75 years. Among a total of 5238 patients who were enrolled in the PROactive study, 2605 patients were randomized to the pioglitazone group and 27 bladder cancer cases were identified finally after doubleblind period +10-year observational follow-up. Meanwhile, 26 cases were reported in the placebo group which consisted of 2633 T2DM patients who took placebo. The authors found no significant association between pioglitazone and bladder cancer with RR and 95% CI 1.05 (0.61-1.79). The other study, TOSCA.IT, was a multicenter prospective, randomized, open label, blinded end point designed. The authors reported eight cases in metformin + pioglitazone (n=1535) and metformin + sulfonylurea group (n=1493). Similar to the PROactive study, no significant relationship was detected. Both studies were assessed to have a low risk of bias [fig_ref] Figure 1: Flowchart of study assessment and selection [/fig_ref].
## Data obtained from observational studies study characteristics
The characteristics of the 12 observational studies are presented in [fig_ref] Table 1: Characteristics of observational studies of pioglitazone and bladder cancer Research Database [/fig_ref]. Of these, eight were cohort studies and four were nested case-control studies. The studies were conducted in the following regions: Europe (n=5), Asia (n=4), and USA (n=3). The study population in 12 studies consisted of both sexes. All included studies were published between 2012 and 2018. The cohort ranged in size from 878 24 to 1.01 million. Medication use in comparison group was either "never used any TZD" or "never used pioglitazone" or "use of sulfonylureas". Adjustments were made for potential confounders of more than two factors in all studies.
## Pioglitazone use and risk of bladder cancer
The overall RR with its 95% CI showed a statistically slight but significant association between pioglitazone use and risk of bladder cancer , RR 1.14, 95% CI 1.03-1.26). There is no significant heterogeneity among these studies with Q=17.86, P=0.085, and I 2 =38.4%.
We also performed dose effect analysis of pioglitazone [fig_ref] Table 2: Dose effect analysis of relative risks for the association between pioglitazone and... [/fig_ref]. We defined low dose as ≤8268 mg or ≤10500 mg or ≤14000 mg; moderate dose as 10501-28000 mg or 14001-40000 mg; high dose as >28000 mg or >40000 mg. Although there were not statistically significant results, the risk of bladder cancer showed a rising trend when the dose of pioglitazone increased (low dose,
# Sensitivity analysis
Sensitivity analyses of the pooled RRs were performed to test the stability of the pooled results. All of the integrated RRs were once again calculated by means of the random effects model. When omitting each study in the meta-analysis, the pooled RRs [fig_ref] Table S3: Sensitivity analysis of relative risks for the association between pioglitazone and bladder... [/fig_ref] always remained stable.
## Publication bias
There was no evidence indicating significant publication bias either with the Begg's test (P=1.0) or with the Egger's test [fig_ref] Figure 3: Egger's publication bias plot [/fig_ref].
# Discussion
The finding of this meta-analysis of 12 studies indicates that pioglitazone is associated with a 14% increased risk of bladder cancer (95% CI 1.04-1.26). Quantified Q test and I 2 test were carried out to assess the extent of heterogeneity among the included studies. No statistically significant heterogeneity was found among the included studies (I 2 =38.4%, P=0.085). Our conclusion was convincing because a fixed model showed a similar result (RR 1.05, 95% CI 1.01-1.10) and our heterogeneity was not significant. In sensitivity analysis we found that heterogeneity largely decreased (I 2 =0, P=0.591) when excluding Levin et al's study. This result may be due to the difference in methodology of Levin et al's paper. Unlike other cohort studies, Levin used a multi-population pooled cohort to assess the risk of bladder cancer and use of pioglitazone in patients with type 2 diabetes. This might be the origin of the heterogeneity. Although the result of two RCT studies providing high-level evidence showed a null association between pioglitazone and bladder cancer, it is still not convincing because a total of only 35 bladder cancer cases were reported in the intervention group. During our search process, another high-quality RCT study assessing risk of bladder cancer and pioglitazone in patients without diabetes was identified. [bib_ref] Pioglitazone after ischemic stroke or transient ischemic attack, Kernan [/bib_ref] Similar to two other RCTs, 12 bladder cancer cases in the pioglitazone group were reported and
## 1632
Yan et al no significant relationship was detected. In spite of its high quality, this study was excluded from the systematic review because the non-diabetic population did not fit our study goal. One important molecular signaling pathway involved in carcinogenesis concerns of pioglitazone intake and risk of bladder cancer. Pioglitazone is a member of TZDs, which are agonists of PPARγ. [bib_ref] The peroxisome proliferator activated receptor gamma agonist pioglitazone increases functional expression of..., Ching [/bib_ref] The effects of PPARγ agonists on human bladder cell lines have continually demonstrated significantly decreased proliferation, increased differentiation and apoptosis, suggesting that PPARγ is involved in antitumor action. [bib_ref] The combinatory effects of PPAR-gamma agonist and survivin inhibition on the cancer..., Wang [/bib_ref] [bib_ref] Role of peroxisome proliferator activated receptor-gamma in bacillus Calmette-Guerin bladder cancer therapy, Langle [/bib_ref] [bib_ref] Effects of PPAR agonists on proliferation and differentiation in human urothelium, Varley [/bib_ref] However, recent research reported by Yang et al indicates that higher expression of PPARγ or its activation by agonist TZD can promote bladder cancer cell migration and invasion, [bib_ref] Higher expression of peroxisome proliferator-activated receptor gamma or its activation by agonist..., Yang [/bib_ref] suggesting that different expression levels of PPARγ may induce different outcomes. In light of this point, pioglitazone, the PPARγ agonist, might cause higher expression of PPARγ which promotes the process of carcinogenesis. In the future, more basic experimental research should be conducted to further elucidate the possible molecular mechanism of pioglitazone intake and risk of bladder cancer.
Our results of dose effect support this molecular theory. Patients who used a higher dose of pioglitazone had an increased risk of bladder cancer, indicating that larger amounts of pioglitazone may cause higher expression of PPARγ which promotes the process of carcinogenesis.
Additionally, no evidence of significant publication bias was observed in the analyses either with the Begg's test or Egger's test. These results greatly improved the predictability and reliability of our meta-analysis. Compared to other related meta-analyses, our metaanalysis evaluating the correlation between pioglitazone and risk of bladder cancer is more convincing because: a) a transparent and robust approach was taken to examine the evidence base, including adherence to PRISMA guidelines; b) we included the newest and the broadest studies, to the best of our knowledge, with rigid inclusion criteria restriction in which only RCTs and high-quality (NOS quality score >6) cohort studies or nested case-control studies were included. We did not include the other high-quality RCT by Kernan et al 47 because the study population excluded patients with type 2 diabetes, which did not fit our aim. One RCT had another quality assessment methodand was systematically reviewed rather than pooling with observational studies. A previous meta-analysis [bib_ref] Pioglitazone prescription increases risk of bladder cancer in patients with type 2..., He [/bib_ref] simply pooled results of RCTs and observational studies which makes the conclusions unconvincing. c) Patients' outcomes were only recorded in reliable databases or using ICD codes. This approach ensured methodological rigor. d) We did not include case-control studies to avoid latent high selection and recall bias; e) previous meta-analyses [bib_ref] An updated meta-analysis of pioglitazone exposure and bladder cancer and comparison to..., Davidson [/bib_ref] [bib_ref] Pioglitazone and the risk of bladder cancer: a meta-analysis, Filipova [/bib_ref] did not exclude studies with duplicate populations. Nevertheless, some limitations should be mentioned: a) we only pooled cohort studies and nested case-control studies without RCTs. Two RCTs, which provided convincing level 1 evidence, reported null association between pioglitazone use and bladder cancer, but there was only a total of 35 bladder cancer cases in the intervention group. More future RCT studies should be performed to provide more convincing evidence. b) Although no publication bias was found in our meta-analysis by either Egger's or Begg's test, the selection strategy of published studies and language limitation could bring about potential publication bias which may have affected our ultimate findings; c) the uncontrolled or unmeasured latent risk factors could have produced biases. We cannot rule out the possibility that remaining confounding factors could have affected the results. Recently, Lewis et al 54 reported that proteinuria testing could potentially cause unmeasured confounding in studies of pioglitazone and bladder cancer but most of the studies included in this metaanalysis 6,7,9,10,12-14 did not adjust for proteinuria testing. d) Although our heterogeneity was not significant, the results of our meta-analysis should be interpreted with caution because heterogeneity of our study was not unapparent (Q=17.86, P=0.085 and I 2 =38.4%).
In conclusion, this meta-analysis indicated that pioglitazone is associated with an increased risk of bladder cancer. Despite some study limitations, we still suggest that doctors should carefully assess the overall risks and benefits of pioglitazone. Further research should be conducted to confirm our findings and clarify the potential biological mechanisms. Cancer Management and Research is an international, peer-reviewed open access journal focusing on cancer research and the optimal use of preventative and integrated treatment interventions to achieve improved outcomes, enhanced survival and quality of life for the cancer patient. The manuscript management system is completely online and includes a very quick and fair peer-review system, which is all easy to use. Visit http://www.dovepress.com/testimonials.php to read real quotes from published authors.
[fig] Figure 1: Flowchart of study assessment and selection. Abbreviations: NOS, Newcastle-Ottawa scale; DM, diabetes mellitus. Cancer Management and Research 2018:10 submit your manuscript | www.dovepress. [/fig]
[fig] Figure 3: Egger's publication bias plot. Notes: Egger's regression asymmetry test (P=0.90). Standardized effect was defined as the odds ratio divided by its standard error. Precision was defined as the inverse of the standard error. Cancer Management and Research 2018:10 submit your manuscript | www.dovepress. [/fig]
[table] Table 1: Characteristics of observational studies of pioglitazone and bladder cancer Research Database; NHI, National Health Insurance; KPNC, Kaiser Permanente Northern California; NHIS-HSC, National Health Insurance Service National Sample Cohort; PSA, Prostate specific antigen. [/table]
[table] Table 2: Dose effect analysis of relative risks for the association between pioglitazone and bladder cancer [/table]
[table] Table S2: Characters of included RCT studiesEvents No events Events No eventsAbbreviations: T2DM, type 2 diabetes mellitus; RCT, randomized controlled trial.Binding of participants and personnel (performance bias)Binding of outcome assessment (detection bias)Figure S1Risk of bias assessment of two RCT studies. Abbreviation: RCT, randomized controlled trial. [/table]
[table] Table S3: Sensitivity analysis of relative risks for the association between pioglitazone and bladder cancer Cancer Management and Research 2018:10 submit your manuscript | www.dovepress.comPublish your work in this journalSubmit your manuscript here: https://www.dovepress.com/cancer-management-and-research-journal [/table]
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Radiation exposure in the remote period after the Chernobyl accident caused oxidative stress and genetic effects in Scots pine populations
Even 30 years after the Chernobyl accident, biological effects of irradiation are observed in the chronically exposed Scots pine populations. Chronic radiation exposure at dose rates above 50 mGy- yr −1 caused oxidative stress and led to the increase of antioxidants concentrations in these populations. Genetic variability was examined for 6 enzymes and 14 enzymatic loci of 6 Scots pine populations. Dose rates over 10 mGy- yr −1 caused the increased frequency of mutations and changes in genetic structure of Scots pine populations. However, the same dose rates had no effect on enzymatic activities. The results indicate that even relatively low dose rates of radiation can be considered as an ecological factor which should be taken into account for ecological management and radiation protection of biota species.As sessile organisms, plants often exist in unfavorable or even stressful conditions, including abiotic and biotic stresses. Such conditions may disturb metabolism, growth and development of plants 1,2 , while studies of plant responses to stress provide important information about the underlying mechanisms of adaptation. The utilization of a stress factor that is easy to measure and which mechanism of action is well-known greatly facilitates the analysis of the adaptation process in natural populations. The ionizing radiation meets both requirements, at least in terms of knowledge of its effects on molecular and cellular levels of biological organization, while an accurate estimation of absorbed dose/dose rate is also possible using specially developed for the experimental object dosimetric models.There are few sites where the influence of ionizing radiation on plant populations in natural conditions can be studied. The Chernobyl accident is known to be the most severe radiation disaster in the human's history. The explosion on 26 th April 1986 contaminated 3 more than 200 000 km 2 with the total released radioactivity of 5 300 PBq 4 . Even now, 30 years after the accident, vast territories remain polluted with radionuclides. Ionizing radiation is a strong mutagenic factor and could possibly have two types of effects: (i) direct effects, which include gene mutations and induction of breaks in DNA by energy transfer; (ii) and indirect effects, which are caused by reactive oxygen species produced by the radiolysis of water molecules. Radiation-induced mutations can alter the DNA structure hence leading to allelic DNA variations. In this context, electrophoresis of isozymes is extremely useful for quantification of the genetic diversity and may be used for assessment of differentiation between populations growing under different ecological conditions. Mutations in isozyme loci are codominant, thus they can be already identified in seeds of the exposed trees 5 . Meanwhile, the evaluation of enzyme activities helps to understand if the high mutational rates in isozyme loci influence on the physiological state of plants 6 .In this study we aimed to understand if relatively low levels of radiation exposure (0.03-66.6 mGy- yr −1 ) cause changes in the genetic structure and antioxidant system of chronically irradiated pine populations. For this purpose we analyzed Scots pine populations that have been growing for 30 years on the heavily contaminated by the Chernobyl accident territories of Russia and Republic of Belarus.Methods and MaterialsTest organism. Scots pine (Pinus sylvestris L.) was chosen as a test organism for an assessment of the possible effects of radioactive contamination. It is the dominant tree species in North European and Asian boreal forest, and is widespread on the area contaminated by the Chernobyl accident. The reproductive organs of conifers are
especially sensitive to radiation exposure because of their complex organization and long generative cycle [bib_ref] Radiobiological and radioecological studies of woody plants, Kozubov [/bib_ref] [bib_ref] The cellular and molecular biology of conifer embryogenesis, Cairney [/bib_ref]. The presence of a haploid endosperm (megagametophyte) in seeds allows direct determination of a haplotype and recessive mutations [bib_ref] Are long-lived trees poised for evolutionary change? Single locus effects in the..., Verta [/bib_ref]. Due to its wide distribution and high radiosensitivity, Scots pine is regarded as one of the basic reference species in the modern concept of the radiation protection of the environment 9 .
Experimental sites. Our experimental sites are located within the area which was significantly contaminated as a result of the Chernobyl accident. There are two reference and four experimental sites in the Bryansk region of Russia [fig_ref] Figure 1: The location of the reference and experimental sites on radioactively contaminated territories [/fig_ref] , and three experimental sites in the territory of Polesskiy Radiation and Ecological Reserve in Republic of Belarus. Two reference sites have been chosen in order to estimate the natural heterogeneity of the experimental populations, and to make it easier to identify if observed changes in the genetic structure and physiological parameters are connected with the level of radiation exposure.
Samples of soil and biological material were taken on each experimental site for estimation of radionuclides and heavy metal concentrations, physical and chemical properties of soils, namely, soil type, pH, humus content, contents of N, P 2 O 5 , K + , Ca 2+ , Mg 2+ , cation exchange capacity, hydrolytic acidity. It was found that in terms of physical and chemical properties of soils and heavy metal pollution our sites are quite similar [bib_ref] Effects of radioactive contamination on Scots pines in the remote period after..., Geras'kin [/bib_ref] , while the level of radioactive contamination significantly varies from site to site . Data received from meteorological stations showed [bib_ref] Specific features of Scots pine seeds formation in the remote period after..., Geras'kin [/bib_ref] that moisture and temperature regimes do not differ essentially among experimental sites. Dose rate assessment. For the dose rate assessment we used data on radionuclides activities ( 137 Cs, Sr, Pu, 241 Am) in soil and pine cones, according to the dosimetric model previously developed for calculation of the total (internal+ external) radiation dose absorbed by pine trees crown Sampling. Soil samples were taken at each experimental site at depths of 0-5, 5-10 and 10-15 cm from 3-4 different points under the pine crowns. The different points were merged into one representative sample from each experimental site. Pine cones were collected during December of 2009-2013 from 30 to 50 years old Scots pine trees in Bryansk region (6 experimental sites). At each site, 30-50 cones were taken from 20-29 trees, at a height of 1.5-2.0 m above the ground. Only freely released and well-formed seeds were used for the electrophoretic analyses. Pine needles were collected in November 2015 at the experimental sites in Bryansk region and in Republic of Belarus (9 experimental sites in total). Samples of needles were obtained from 15-18 trees at each experimental site. Five needles were harvested from different sides of each tree, no more than 2.5 m above the ground level. The needles were placed into cryovials and frozen in liquid nitrogen until analysis.
Electrophoretic analysis of enzyme polymorphism. Six enzymes were chosen for the study of genetic diversity and mutational rates in the experimental populations. Three of them represent the antioxidant system, which is expected to show strong reaction to chronic radiation exposure: superoxide dismutase (EC 1.15.1.1, SOD), glutathione reductase (EC 1.6.4.2, GR), and glutathione peroxidase (EC 1.11.4.2, GPX) [bib_ref] Genetic diversity in Scots pine populations along a radiation exposure gradient, Geras'kin [/bib_ref]. The other three represent different parts of anabolic-catabolic pathways, which are expected to react less evident to chronic radiation exposure, because stability of these systems has high importance for an individual survival: malate dehydrogenase (EC 1.1.1.37, MDH), glucose-6-phosphate dehydrogenase (EC 1.1.1.49, G6PD) and leucine aminopeptidase (EC 3.4.11.1, LAP). The analysis was carried out using 15 endosperms per tree in average. Seeds from different sites were randomized and encoded. Each endosperm was individually homogenized in 100 μ l of the extraction buffer (1% triton Х− 100 solution and 0.2% solution of β -mercaptoethanol). Supernatants were used for enzyme assays after homogenization and centrifugation (14,500 rpm for 10 min).
Electrophoresis was carried out in vertical chambers «Protean II xi Cell» (USA) and «Hoefer SE 600 Chroma» (USA), using a 7.5% polyacrylamide gel in a Tris-HCl buffer system рН 8.0 with Tris-glycine pH 8.9 as an electrode buffer, for 1.5-2.0 h at 60-80 mA. Gel polymerization took 40-60 min. Isozymes were stained by conventional techniques. Only those bands that could be scored without ambiguity were taken into account. In total, 13,116 loci tests were performed.
Three types of radiation-induced mutations were revealed. Null mutations were identified as an absence of the corresponding allelic variant in the gel. Duplications were manifested by appearance of two enzyme bands in one locus on the gel. Changes in electrophoretic mobility of the isozyme were identified as appearance of the enzyme bands outside the previously identified areas of activity.
## Concentrations of antioxidants.
For isolation of the low-molecular weight antioxidants (LMWA) ascorbic acid (AsA), reduced glutathione (GSH), and oxidized glutathione (GSSG), five needles from each experimental tree were grinded with mortar and pestle in liquid nitrogen. The fine powder was then transferred in 1 ml of cold extraction solution (5% H 3 PO 4 , 1 mM EDTA in 0.1% formic acid). The homogenates were centrifuged at 14,500 rpm for 20 min under 4 °C. The supernatants were collected, while the pellets were resuspended with 1 ml of the same extraction solution and centrifuged again under the same conditions. The second supernatant was combined with the first one and filtered through Whatman 0.2 μ m nylon membranes with glass microfiber prefilter. For each sample, an aliquot of 500 μ l from this mix was stabilized by the addition of 50 μ l of 2% butylated hydroxytoluene in ethanol [bib_ref] High-throughput determination of malondialdehyde in plant tissues, Davey [/bib_ref] and stored at − 20 °C for the analysis concentration of the oxidative stress reporter malondialdehyde (MDA). The LMWA of the remaining mix was immediately analyzed. All steps were performed at 4 °C with two technical replicates for each sample.
Analyses of LMWA and MDA concentrations were carried out with a high-performance liquid chromatography system Shimadzu LC-30 (Shimadzu, Japan). The analyses were done by injecting of 10 μ l aliquots of standard solutions and sample extracts in a reverse-phase columns Shim-pack XR-ODSII (Shimadzu, 2.2 μ m, 3.0 × 100 mm, Japan). For LMWA, the autosampler and column temperatures were 6 °C and 30 °C, respectively, while 6 °C and 40 °C were used for MDA. Samples were eluted at a flow rate of 0.5 ml/min. For LMWA estimation the mobile phase was built using two solvents: solvent A (0.1% formid acid in Milli-Q water) and B (0.1% formic acid in acetonitrile) [bib_ref] Direct and simultaneous determination of reduced and oxidized glutathione and homoglutathione by..., Rellán-Álvarez [/bib_ref]. For separation of the analytes we used a linear gradient of B from 0 to 10% (0-7 min). Then the column was washed by a linear increase of B concentration from 10 to 90% from 7 to 9 min, being this solvent composition used until 10 min. For the column regeneration, the solvent composition was changed linearly to 0% of B until 12 min, being then was maintained in this state until 17 min. After that a new sample could be injected. Retention times for analytes were 1.7 min (265 nm) for ascorbic acid, 2.1 min (214 nm) for reduced and 3.3 min (214 nm) for oxidized glutathione.
For MDA identification we also used two solvents [bib_ref] High-throughput determination of malondialdehyde in plant tissues, Davey [/bib_ref] : solvent A (50 мМ KH 2 PO 4 in MilliQ water) and B (methanol). For this analysis we used linear gradient from 0-10% of B (0-3 min). Then the column was washed with linear gradient of B from 10 to 90% (3-5 min), being this solvent composition maintained until 6 min. For the column regeneration step the concentration of solvent B was reduced from 90 to 0% (6-8 min), while 100% of A solvent was used until the 11th min. Retention time for MDA standard was 2.4 min (267 nm).
Validation for both methods was provided by obtaining calibration curves, by identification of peaks identities with internal standards, and by estimation of limits of detection. The HPLC system works under control of the software package Lab Solutions (Shimadzu).
Enzymatic activities analysis. Activities of enzymes (superoxide dismutase, catalase, guaiacol peroxidase, malate dehydrogenase, glucose-6-phosphate dehydrogenase, leucine aminopeptidase) in pine endosperms were assessedusing «NanoDrop-2000» spectrophotometer (USA).
Statistical analysis. The data are presented as «mean value ± SE». Significance of difference between the means was determined using the t-test. The normal distribution of experimental data was checked prior the statistical analysis. Correlation analysis was done using the Pearson correlation coefficient. We did not apply the multiple comparisons due to using only 2 reference and 7 experimental conditions in our experiment, so its design is different from "case-control" design.
For each population we calculated the allele frequencies and number of indices, which reflect the genetic diversity in experimental populations. An index of the allelic diversity μ and its error s μ were calculated 17 using (1, 2):
[formula] μ = + +… + ( ) p p p (1) m 1 2 2 and µ µ = − µ s m N ( )(2) [/formula]
where p 1 , p 2 , … p m are the frequencies of alleles, m is the number of alleles, N -the sample size. Effective number of alleles (n e ) was determined as follows (3) 17 :
[formula] ∑ = е n 1/ p (3) i 2 [/formula]
Genetic distances (D) between populations were evaluated as follows (4) 18 :
[formula] = − = − + + ( ) D r J J J ln ln 1 2 ln ln ,(4)pq p q [/formula]
where J p is the averaged over all loci theoretical homozygosity in the first population, J q is the averaged over all loci theoretical homozygosity in the second population,.. is the mutual identity of two populations for all loci. The genetic distances matrix was analyzed using cluster analysis by applying the unweighted pair group method (UPGMA) in order to group the populations based on their genetic similarity. Statistical analysis was done using MS Office Excel 2007 software and Statistica 8.0 for Windows.
# Results
The absorbed doses. The doses annually absorbed by the pine crowns should be regarded as low.
However, one should remember that experimental trees had received much higher doses during the first year after the Chernobyl accident, mainly from short-lived radionuclides [bib_ref] An assessment of cumulative external doses from Chernobyl fallout for a forest..., Ramzaev [/bib_ref].
Isozyme polymorphism. The isozyme analysis revealed an increased mutational rate in chronically irradiated populations . The frequency of mutations has shown strong correlation with the level of radiation exposure at the experimental sites (r 2 = 0.98, p < 0.001).
All detected loci and allele frequencies of the six evaluated enzymes are presented in . Indices of genotypic diversity [fig_ref] Figure 2: Characteristics of genetic structure of chronically exposed pine populations [/fig_ref] and effective numbers of alleles [fig_ref] Figure 2: Characteristics of genetic structure of chronically exposed pine populations [/fig_ref] were calculated for two groups of enzymes. Anabolic and catabolic enzymes are characterized by significantly higher genotype diversity and effective numbers of alleles in comparison with antioxidant enzymes. Considering this, we estimated the mean values of the characteristics of genetic structure of populations separately for each group of enzymes. Interestingly, genotypic diversity of antioxidant enzymes is significantly higher at the most contaminated sites, and shows strong dependence on the absorbed dose [fig_ref] Figure 2: Characteristics of genetic structure of chronically exposed pine populations [/fig_ref] Based on the information about presence and frequencies of alleles, the analysis of genetic distance between experimental populations showed two well-defined groups: the first one included the populations inhabiting heavily contaminated sites (Z1 and Z2), and the second one included much less contaminated sites (Ref, Ref1, VIUA, and SB) [fig_ref] Figure 3: The diagram of genetic distances among 6 experimental populations from radioactively contaminated... [/fig_ref].
Enzyme activities. Information about biological activity of enzymes is essential for understanding how a gene function and a genetic variation affect phenotypes [bib_ref] Why measure enzyme activities in the era of systems biology?, Stitt [/bib_ref]. In this experiment, significant changes in the activities were found only for POD and G6PD [fig_ref] Table 3: Antioxidant concentrations and enzyme activities in chronically irradiated Scots pine populations [/fig_ref]. The activities of the remaining four enzymes (SOD, CAT, MDH, and LAP) did not depend on the absorbed dose at all (r 2 = 0.01-0.45, p < 0.05). While POD activity decreased with the dose rate (r 2 = 0.79, p < 0.01), the activity of G6PD increased (r 2 = 0.77, p < 0.01).
## Low-weight antioxidant molecules quantification.
Concentrations of low molecular weight antioxidants did not show [fig_ref] Table 3: Antioxidant concentrations and enzyme activities in chronically irradiated Scots pine populations [/fig_ref] any significant dependence on the overall level of radiation exposure (r 2 = 0.20, p > 5% for GSH, r 2 = 0.20, p > 5% for GSSG, r 2 = 0.02, p > 5% for AsA). Nevertheless, GSH concentration significantly increased at the most contaminated sites, while GSSG showed an inverse pattern. More interesting results are revealed by the analysis of the GSH/GSSG ratio, which is essentially higher for the most contaminated sites and shows significant correlation (r 2 = 0.446, p < 0.05) with the level of radiation exposure. Analysis of MDA concentrations showed that experimental populations have undergone the oxidative stress (r 2 = 0.397, p < 0.05), which is especially evident for the most exposed population Kul [fig_ref] Table 3: Antioxidant concentrations and enzyme activities in chronically irradiated Scots pine populations [/fig_ref].
# Discussion
Currently, international organizations are developing a new approach for radiation protection of biota species.
There is yet a sufficient lack of field data about the real consequences of chronic radiation exposure for the plant and animal populations [bib_ref] Are radiosensitivity data derived from natural field conditions consistent with data from..., Garnier-Laplace [/bib_ref]. The existing estimates are quite inaccurate and are often based on scientific consensus, while the real experimental data from natural conditions are needed for development of the effective management strategy for radioactively polluted areas. For instance, United Nations Scientific Committee on the Effects of Atomic Radiation considers 100 μ Gy- h −1 as safe dose rate for plant ecosystems 21 , but even significantly lower dose rates in our study already induce the number of biological effects in populations of Scots pine -the species that can be considered as the main and the most radiosensitive component of a coniferous forest ecosystem [fig_ref] Table 3: Antioxidant concentrations and enzyme activities in chronically irradiated Scots pine populations [/fig_ref] , [fig_ref] Figure 2: Characteristics of genetic structure of chronically exposed pine populations [/fig_ref]. Our results suggest that the effective radiation protection approach should be based on the information about effects on populations of reference species and should include different dose thresholds for different types of reference ecosystems. The studied Scots pine populations have been growing under chronic radiation exposure for almost 30 years. Initially, the levels of radioactive contamination at the experimental sites were high enough [bib_ref] An assessment of cumulative external doses from Chernobyl fallout for a forest..., Ramzaev [/bib_ref] to induce strong biological effects because of the short-lived radioisotopes. Observed biological effects in our study most likely have two reasons: initial high-dose irradiation, and the long-term chronic exposure, which diminishes each passing year. Both of them could probably result in differential gene expression patterns of irradiated individuals. In this context, our experimental sites provide the unique opportunity to study the initial steps of genetic differentiation in natural populations under severe stressful conditions. Changes in genetic structures have been detected in response to strong environmental stress for a number of plant species including forest trees: industrial pollution for Pinus sylvestris [bib_ref] Industrial pollutants tend to increase genetic diversity: evidence from field-grown European Scots..., Prus-Glowacki [/bib_ref] and Picea abies [bib_ref] Genetic effects of air pollution on forest tree species of the Carpathian..., Longauer [/bib_ref] ; water and nutrient stress for Pinus edulis [bib_ref] Genetic variation in pinyon pine, Pinus edulis, associated with summer precipitation, Mitton [/bib_ref] ; ozone for Pinus ponderosa [bib_ref] Genetic differences of Pinus Ponderosa [Dougl. ex laws.] trees tolerant and sensitive..., Staszak [/bib_ref] ; temperature for Betula pendula [bib_ref] Temperature based population segregation in birch, Kelly [/bib_ref]. It is known that high level of radiation exposure can also potentially lead to rapid changes in the genetic structure of plant and animal populations [bib_ref] Integration of genotoxic and population genetic endpoints in biomonitoring and risk assessment, Theodorakis [/bib_ref] , and in our study we consider the increased mutational rate as a potential engine of genetic changes in chronically irradiated pine populations . We discovered that some indices of genetic diversity were increasing along the level of radiation exposure [fig_ref] Figure 2: Characteristics of genetic structure of chronically exposed pine populations [/fig_ref] and frequencies of different alleles significantly varied from site to site (Supplementary . Genetic differentiation of experimental populations apparently is driven by radiation exposure, especially in the heavy contaminated sites [fig_ref] Figure 3: The diagram of genetic distances among 6 experimental populations from radioactively contaminated... [/fig_ref]. Two studied groups of enzymes showed different response to radiation exposure. Antioxidant enzymes play an important role in the ability of population to withstand oxidative stress, induced by chronic irradiation. Possible variations in their allelic structure may be less risky for population as the alterations in the allelic structure of anabolic and catabolic enzymes. Indeed, the genotypic diversity of antioxidant group on the contaminated sites rises faster in comparison with MDH, G6PD and LAP, which even showed a decrease of genotypic diversity at the less contaminated areas in comparison with reference sites. Effective number of alleles, as expected, is higher in the group of anabolic and catabolic enzymes, because loci of these enzymes have very high allelic variability even in the reference groups. However, there were no significant changes in the activities of six evaluated enzymes [fig_ref] Table 3: Antioxidant concentrations and enzyme activities in chronically irradiated Scots pine populations [/fig_ref] that suggests that the dose rate in the range of 0.03-38.6 mGy- yr −1 is insufficient to induce an essential biological effect at this level of organization.
The analysis of antioxidants and MDA concentrations showed that even low-dose chronic radiation exposure contributes to the oxidative stress in natural populations [fig_ref] Table 3: Antioxidant concentrations and enzyme activities in chronically irradiated Scots pine populations [/fig_ref]. MDA, which is a decomposition product of polyunsaturated fatty acids in membranes, is considered to be an indicator of free radical stress. The increase of MDA concentrations indicates a distinct deterioration of membrane integrity and activation of lipid peroxidation in plant cells. The MDA concentrations are increased at radioactively contaminated sites and show slight dependence on the level of radiation exposure [fig_ref] Table 3: Antioxidant concentrations and enzyme activities in chronically irradiated Scots pine populations [/fig_ref]. In spite of the increased levels of MDA, our study did not reveal any significant changes in the free AsA concentrations in the affected plant populations [fig_ref] Table 3: Antioxidant concentrations and enzyme activities in chronically irradiated Scots pine populations [/fig_ref]. The ascorbate concentration fluctuates depending on many factors such as light intensity, age, plant tissue, and cell compartment [bib_ref] Ascorbate biosynthesis in mitochondria is linked to the electron transport chain between..., Bartoli [/bib_ref]. Probably, more detailed investigation of reduced and oxidized forms of AsA and their ratio in the experimental populations may give more valuable information about the role of this antioxidant in response to chronic low-dose radiation exposure.
Glutathione is an important component of the defense system in plants and animals. Under stress it accumulates in cells, protecting them to oxidative stress by reducing H 2 O 2 . Glutathione is therefore a key factor for cellular redox homeostasis and tolerance against abiotic and biotic stresses, and mainly exists in two forms -reduced (GSH) and oxidized (GSSG) glutathione. The accumulation of glutathione and other antioxidants is commonly observed in plants under stress, as a protective response that may determine whether stress causes only slight and temporary deviations from the normal state, or more severe and permanent damage [bib_ref] Glutathione and glutathione reductase: a boon in disguise for plant abiotic stress..., Gill [/bib_ref]. The changes of the ratio GSH/GSSG during the degradation of H 2 O 2 is important in certain redox signaling pathways [bib_ref] Common components, networks and pathways of cross-tolerance to stress. The central role..., Pastori [/bib_ref]. Increased glutathione concentrations and GSH/GSSG ratio at the most contaminated sites can be considered as an adaptive reaction. Therefore, the increase of GSH concentration may lead to enhancement of stress tolerance in the chronically irradiated plant populations.
The connection between isozyme polymorphism and an activity of antioxidant system in plants under stress conditions was shown in a few works [bib_ref] Superoxide dismutase activity and antioxidant response of hydroponically cultured Lepidium sativum L...., Manaa [/bib_ref] [bib_ref] Copper-stress induced alterations in protein profile and antioxidant enzymes activities in the..., Rout [/bib_ref]. It suggests that some changes observed in the antioxidant system may be due to alterations in the genetic structure of studied populations. In the same time, the main difficulty of our work is to separate the effects of primary high-dose irradiation and of current chronic low-dose exposure. We assume that at least some effects (the increased rate of null-mutations and the high level of oxidative stress at the most contaminated sites) could be connected with the present levels of chronic exposure. Our previous work showed 33 that the spectrum of chromosomal abnormalities at the most contaminated sites (chromosome lagging, multipolar mitoses) suggested their radiation nature. The maintenance of high MDA concentrations also gives evidence of the heightened concentrations of reactive oxygen species in plant cells, which probably provoke the response of the antioxidant system showed in this research.
Natural ecosystems are often polluted with a mixture of pollutants that can cause a synergetic influence of different stressors on an ecosystem. Different biotic and abiotic factors may interact with pollutants, also causing synergetic effects. A number of works showed synergetic interaction between ionizing radiation and heavy metals: γ -radiation and Cr 34 , γ -radiation and Ni/As 35 , γ -radiation and Cd/Pb 36 . Based on knowledge of possible coaction of ionizing radiation and heavy metals, we carefully chose the experimental sites and made sure that there was no excess of heavy metal concentrations in soil. Another possible input in the observed biological effect could be made by weather conditions and UV-radiation, which is known to cause DNA-damage and interacts synergistically with ionizing radiation [bib_ref] The synergistic action of ultraviolet and X-radiation on mutants of Escherichia coli..., Martignoni [/bib_ref]. To eliminate this factor the experimental sites were chosen taking into account weather conditions; moreover, the multiple regression analysis of different weather factors (i.e. humidity, the average temperatures, the amount of precipitation etc) did not show any interaction of them with the level of radiation exposure at the experimental sites [bib_ref] Specific features of Scots pine seeds formation in the remote period after..., Geras'kin [/bib_ref].
Overall, this study has shown that it is important to estimate the reaction of living things to anthropogenic exposure using different approaches at different levels of biological organization. Strong biological effects at the molecular level of organization turn into moderate effects at the physiological level and into slight effects at the organismal level [bib_ref] Effects of radioactive contamination on Scots pines in the remote period after..., Geras'kin [/bib_ref] [bib_ref] Study of needles morphometric indexes in Scots pine in the remote period..., Makarenko [/bib_ref]. The effect of low-dose chronic irradiation on individuals has remained a matter of discussions, but from the standpoint of our research even quite low doses of radiation exposure influence on a genetic structure of natural population through an increase in the mutational rates. Higher dose rates (starting from 51 mGy- yr −1 in this research) already maintain a higher level of oxidative stress and cause the response of antioxidant system. Recently we have started a new experiment on the first generation of irradiated pine trees (non-exposed in the acute period of disaster, age < 25 years), evaluating gene expression profiling. We hope that this data will help to separate biological effects of acute exposure from chronic exposure effects.
# Conclusions
Studies of the mechanisms of adaptation to environmental stress create a scientific basis for developing the strategy of biota protection from man-made impact. The present work is a part of the continuing long-term (2003-2016) study of the biological effects in Scots pine populations, showing that affected Scots pine populations are characterized by an increased level of cytogenetic abnormalities in root meristem of seedlings [bib_ref] Effects of radioactive contamination on Scots pines in the remote period after..., Geras'kin [/bib_ref]. However, the higher rate of mutations had no effect on the reproductive ability of these trees, which is largely determined by weather conditions [bib_ref] Specific features of Scots pine seeds formation in the remote period after..., Geras'kin [/bib_ref]. It follows from this work that relatively low levels of radiation exposure (up to 39 mGy- yr −1 ) do not significantly change studied physiological parameters in Scots pine populations, but they lead to the occurrence of higher mutational rates and changes in the genetic structure of populations in comparison with the reference sites. At the most contaminated experimental sites (51-66 mGy- yr −1 ), with the higher contribution of β -radiation to annual dose, we observe difference in the antioxidant status and higher level of oxidative stress compared to the reference sites. Consequently, plant populations remain a great object for study of long-term biological consequences of radiation exposure even 30 years after the Chernobyl accident.
[fig] Figure 1: The location of the reference and experimental sites on radioactively contaminated territories. 1 -Ref, 2 -Ref1, 3 -Z1, 4 -Z2, 5 -VIUA, 6 -SB, 7 -Kul, 8 -Mas, 9 -Kozh. The map was created using Google Maps service (the attribution can be seen in the bottom right corner of the Figure) and modified in CorelDraw Graphics Suite X7. Levels of radioactive contamination (in 1998) according to ref. 3. [/fig]
[fig] 2: = 0.94, p < 0.01). Genotypic diversity of anabolic and catabolic enzymes shows much weaker correlation with radiation exposure (Fig. 2B; r 2 = 0.65, p > 5%). On the other hand, the effective number of alleles for antioxidant enzymes had weak correlation with the dose rate Scientific RepoRts | 7:43009 | DOI: 10.1038/srep43009 (Fig. 2C; r 2 = 0.66, p > 5%), while anabolic and catabolic enzymes showed stronger correlation with this value and radiation exposure (Fig. 2D; r 2 = 0.81, p < 0.01). [/fig]
[fig] Figure 3: The diagram of genetic distances among 6 experimental populations from radioactively contaminated territories of Russia. [/fig]
[table] Table 3: Antioxidant concentrations and enzyme activities in chronically irradiated Scots pine populations. n/e -not evaluated; RF Russian Federation; RB Republic of Belarus. t-test revealed a significant difference with: *Ref site, p < 0.05; ***Ref site, p < 0.001; ▲ Ref1 site, p < 0.05; ▲▲ Ref1 site, p < 0.01; ▲▲▲ Ref1 site, p < 0.001. RepoRts | 7:43009 | DOI: 10.1038/srep43009 [/table]
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Helium Generated Cold Plasma Finely Regulates Activation of Human Fibroblast-Like Primary Cells
Non-thermal atmospheric pressure plasmas are being developed for a wide range of health care applications, including wound healing. However in order to exploit the potential of plasma for clinical applications, the understanding of the mechanisms involved in plasma-induced activation of fibroblasts, the cells active in the healing process, is mandatory. In this study, the role of helium generated plasma in the tissue repairing process was investigated in cultured human fibroblast-like primary cells, and specifically in hepatic stellate cells and intestinal subepithelial myofibroblasts. Five minutes after treatment, plasma induced formation of reactive oxygen species (ROS) in cultured cells, as assessed by flow cytometric analysis of fluorescence-activated 29,79-dichlorofluorescein diacetate probe. Plasma-induced intracellular ROS were characterized by lower concentrations and shorter half-lives with respect to hydrogen peroxide-induced ROS. Moreover ROS generated by plasma treatment increased the expression of peroxisome proliferator activated receptor (PPAR)-c, nuclear receptor that modulates the inflammatory responses. Plasma exposure promoted wound healing in an in vitro model and induced fibroblast migration and proliferation, as demonstrated, respectively, by trans-well assay and partitioning between daughter cells of carboxyfluorescein diacetate succinimidyl ester fluorescent dye. Plasma-induced fibroblast migration and proliferation were found to be ROS-dependent as cellular incubation with antioxidant agents (e.g. N-acetyl L-cysteine) cancelled the biological effects. This study provides evidence that helium generated plasma promotes proliferation and migration in liver and intestinal fibroblast-like primary cells mainly by increasing intracellular ROS levels. Since plasma-evoked ROS are time-restricted and elicit the PPAR-c anti-inflammatory molecular pathway, this strategy ensures precise regulation of human fibroblast activation and can be considered a valid therapeutic approach for liver and gut lesions. Citation: Brun P, Pathak S, Castagliuolo I, Palù G, Brun P, et al. (2014) Helium Generated Cold Plasma Finely Regulates Activation of Human Fibroblast-Like Primary Cells. PLoS ONE 9(8): e104397.
# Introduction
Non-thermal atmospheric-pressure plasmas have recently been investigated for multiple medical applications [bib_ref] Plasma medicine: an introductory review, Kong [/bib_ref]. Besides sterilizing inert surfaces [bib_ref] Effect of gas composition on spore mortality and etching during low-pressure plasma..., Lerouge [/bib_ref] , disinfecting living tissue [bib_ref] Disinfection of ocular cells and tissues by atmospheric-pressure cold plasma, Brun [/bib_ref] [bib_ref] Decolonisation of MRSA, S. aureus and E. coli by cold-atmospheric plasma using..., Maisch [/bib_ref] , disposing of cancer cells and bleeding control [bib_ref] Targeting cancer cells with reactive oxygen and nitrogen species generated by atmosphericpressure..., Ahn [/bib_ref] [bib_ref] Lowtemperature plasma-induced antiproliferative effects on multi-cellular tumor spheroids, Plewa [/bib_ref] [bib_ref] Formation of Membrane-like Structures in Clotted Blood by Mild Plasma Treatment during..., Ikehara [/bib_ref] , cold plasmas of various gas compositions have been found to promote wound healing and tissue regeneration [bib_ref] Randomized placebo-controlled human pilot study of cold atmospheric argon plasma on skin..., Heinlin [/bib_ref] [bib_ref] Accelerated mice skin acute wound healing in vivo by combined treatment of..., García-Alcantara [/bib_ref] [bib_ref] Differential influence of components resulting from atmospheric-pressure plasma on integrin expression of..., Haertel [/bib_ref]. Nevertheless plasma functionalization of medical implant devices enhances the attachment and spreading of cells in vivo, suggesting that plasma can also interact with the extracellular environment shaping the properties of surface proteins [bib_ref] Osseointegration assessment of chairside argon-based nonthermal plasma-treated Ca-P coated dental implants, Giro [/bib_ref] [bib_ref] Plasma treatment for improving cell biocompatibility of a biodegradable polymer scaffold for..., Sd [/bib_ref]. Most of plasma's effects are strictly linked to reactive oxygen species (ROS) production [bib_ref] Disinfection of ocular cells and tissues by atmospheric-pressure cold plasma, Brun [/bib_ref] [bib_ref] Targeting cancer cells with reactive oxygen and nitrogen species generated by atmosphericpressure..., Ahn [/bib_ref]. Indeed, ROS generated during plasma exposure could be responsible for lipid peroxidation of membrane in bacteria [bib_ref] Nonthermal dielectric-barrier discharge plasma-induced inactivation involves oxidative DNA damage and membrane lipid..., Joshi [/bib_ref] , releasing growth factors in proliferating cells [bib_ref] Endothelial cell proliferation is enhanced by low dose non-thermal plasma through fibroblast..., Kalghatgi [/bib_ref] , and DNA damage in cancer cell lines [bib_ref] Lowtemperature plasma-induced antiproliferative effects on multi-cellular tumor spheroids, Plewa [/bib_ref] [bib_ref] ROS implication in a new antitumor strategy based on non-thermal plasma, Vandamme [/bib_ref]. In the gas phase, non-thermal plasmas contain neutral non-charged species which, following interaction with organic cellular components as well as media enriched in specific amino acids, lead to the production of excited molecules, ions, and radicals [bib_ref] Effects of non-thermal plasma on mammalian cells, Kalghatgi [/bib_ref]. This is why some concerns have been expressed with regard to employing plasma in in vivo medical treatments and in wound healing. For example, non-thermal atmospheric pressure plasma affects the expression of adhesion molecules on cellular surfaces causing loss of cell-cell interaction and cellular detachment in HaCaT-keratinocytes [bib_ref] Non-thermal atmospheric-pressure plasma can influence cell adhesion molecules on HaCaT-keratinocytes, Haertel [/bib_ref]. Despite a large body of scientific evidence that has been collected with regard to immortalized cell lines, few studies have been performed until now to investigate the effects of plasmas on cell adhesion and proliferation of primary fibroblasts [bib_ref] Cold atmospheric plasma (CAP) changes gene expression of key molecules of the..., Arndt [/bib_ref] , the main cell population contributing to the healing process.
Wound healing is a dynamic, well-organized process requiring the cooperation of different cell types to repair skin lesions and damaged internal organs [bib_ref] Wound repair and regeneration, Gurtner [/bib_ref]. At first, low local levels of cytokines, chemokines, and components of the renin-angiotensin system trigger proliferation and migration of mesenchymal and nonmesenchymal cells to promote wound healing. But the critical event in wound healing is the transition of tissue resident cells to activated fibroblasts which produce growth factors and a collagen framework that promote tissue regeneration [bib_ref] Pathogenesis of liver fibrosis, Hernandez-Gea [/bib_ref] [bib_ref] Role of intestinal subepithelial myofibroblasts in inflammation and regenerative response in the..., Andoh [/bib_ref]. One of several mediators, ROS regulate myofibroblast activation and play a key role in extracellular matrix deposition [bib_ref] Role of hydrogen peroxide and oxidative stress in healing responses, Rojkind [/bib_ref]. Thus, upon toxic insult or viral infection, reactive oxygen intermediates are mandatory to activate and differentiate quiescent hepatic stellate cells (HSCs) into contractile myofibroblast-like cells, the main source of extracellular matrix components during wound healing responses in the liver [bib_ref] The role of redox mechanisms in hepatic chronic wound healing and fibrogenesis, Novo [/bib_ref]. Threshold levels of ROS are likewise critical in maintaining epithelial restitution, reconstitution, and barrier function in intestinal subepithelial myofibroblasts (ISEMFs), whereas increased ROS levels have been linked to gut fibrosis [bib_ref] IL-1alpha-induced COX-2 expression in human intestinal myofibroblasts is dependent on a PKCzeta-ROS..., Mari [/bib_ref]. The physiological wound healing process is, however, often jeopardized by intrinsic and environmental factors. Indeed, infiltrating leukocytes, neighbouring damaged cells, and/or microbes infecting the wound increase inflammatory cytokine levels and exaggerate ROS generation thus perpetuating myofibroblast activation with excessive accumulation of collagen and fibrotic scarring [bib_ref] Oxidative and nitrosative stress and fibrogenic response, Urtasun [/bib_ref] [bib_ref] Mechanisms of hepatic fibrogenesis, Friedman [/bib_ref].
We recently reported that atmospheric pressure non-thermal plasma needle generated by ionizing helium gas slightly increased intracellular ROS levels in primary human keratocytes and conjunctival fibroblasts [bib_ref] Disinfection of ocular cells and tissues by atmospheric-pressure cold plasma, Brun [/bib_ref]. The aim of the present study was to investigate the effects of plasma-induced ROS on the proliferation and migration of cultured human primary HSCs and ISEMFs and to specifically assess if controlling myofibroblast activation and modulating their pro-fibrogenic effects can accelerate the wound healing without fibrotic scar formation.
# Materials and methods
## Cell culture
Human HSCs were freshly isolated from non-pathological fragments of liver tissue collected from 3 patients during surgical resection of liver metastases. The samples were processed separately and HSCs were cultured as described elsewhere [bib_ref] Exposure to bacterial cell wall products triggers an inflammatory phenotype in hepatic..., Brun [/bib_ref]. Briefly, following digestion with collagenase and pronase, HSCs were isolated by centrifugation over a gradient of Percoll (Amersham Biosciences, Sweden) and cultured in DMEM containing 10% vol/vol fetal bovine serum (FBS), 2 mM Lglutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin, (all provided by Gibco, Italy). The purity of cultured HSCs was assessed by immunocytochemistry using anti-aSMA antibody (Sigma, Italy).
ISEMFs were isolated from non-pathological colonic biopsies collected from 3 subjects undergoing colonoscopy for cancer screening. The tissue samples were diced, extensively washed, and digested for 30 min at 37uC in collagenase (0.25 mg/ml, Sigma). Recovered cells were suspended in DMEM with 20% vol/vol FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 100 U/ml penicillin, 100 mg/ml streptomycin and 2 ng/mL fungizone (Gibco). The purity of the cultured ISEMFs was ascertained by fluorescence-activated cell sorting (FACS) analysis using anti-CD90 antibody (ImmunoTools, Germany). Primary cells were maintained at 37uC in a 5% CO 2 humidified incubator. At confluence, the cells were detached using 0.05% Trypsin-EDTA (Gibco).
The study protocol was designed in accordance with the principles expressed in the Declaration of Helsinki and was approved by the Ethical Committee of the University Hospital of Padova. Patients were provided with detailed information about the study aims and protocol and gave their written, informed consent.
## Atmospheric pressure cold plasma
Cultured cells were exposed to the effluent coming from a plasma (ionized gas) source specifically designed to treat living tissues. The plasma was produced by applying a radiofrequency (RF) electric field to a flow of helium at atmospheric pressure. The source operated at a very low power level so that the resulting plasma was characterized by a low fraction of ionized particles (ions and electrons). The neutral gas fraction and the ion flow were at room temperature. The electron population was at a temperature to the order of 1 eV (11600 K). Generally called ''cold plasma,'' this plasma source is considered suitable for treating biological samples without thermal effects. The interaction of the electron population with the surrounding air mixed to the helium flow stimulates the production of active chemicals such as ROS, atomic oxygen and hydroxyl radical, and probably accounts for its biological effects.
Plasma was generated between two grids acting as electrodes, and the cultured cells were exposed to the so-called afterglow, which is the chemical-enriched helium flow. In contrast to other cold plasma devices used in biomedical applications, the plasmainduced charged species did not have a direct biological effect on the cultured cells since they recombined very quickly before reaching the samples. This plasma source has been described elsewhere [bib_ref] A novel plasma source for sterilization of living tissues, Martines [/bib_ref] and is schematically outlined in . As indicated, the source consists of two coaxial tubes, each closed at one end by a double brass grid. The outer tube is made out of copper and electrically grounded while the inner one is made out of insulating material. The two parallel grids are positioned 1 mm away from one other. An electric field is formed in the space between the two grids by applying a RF voltage difference supplied by a RF generator coupled to the source by a matching network. The matching network raises the voltage to the value needed for helium ionization of about 1000 V peak-to-peak. Despite the high voltage value, the current flowing in the plasma is so low that the dissipated power is below 1 W. The chosen operational frequency is 4.8 MHz. The gas flow rate is 1.5 litres/ min.
## Detection of intracellular ros levels
HSCs and ISEMFs were seeded in 24-well tissue culture plates (Corning, Italy) at 3610 5 cells/ml to assess ROS generation. Twenty-four hrs later cells were loaded for 30 min at 37uC with 10 mM 29,79-dichlorodihydrofluorescein diacetate (H 2 DCFDA; Molecular Probes, Invitrogen, Italy) in warm PBS [bib_ref] Disinfection of ocular cells and tissues by atmospheric-pressure cold plasma, Brun [/bib_ref]. At the end of the incubation, cultures were washed twice, incubated with 200 ml of fresh serum-free culture medium, and exposed for 1, 2, 5, or 10 min to atmospheric pressure cold plasma. The tip of the plasma source was positioned within 1.50 to 2.40 mm from the surface of the medium soaking the samples. The cells were then incubated at 37uC for 5 min-24 hrs, harvested by Trypsin-EDTA, washed and analyzed using BD FACS-Calibur flow cytometer (Becton Dickinson, USA). Ten thousands events were acquired for each sample. The results were analyzed using the WinMDI 2.
## Mitochondrial dysfunction
Plasma treated cells or cells stimulated with H 2 O 2 were incubated for 1, 6, 24, 48 hrs at 37uC. At the end of the incubation, the cells were collected and re-suspended in PBS containing 1 mM JC-1 (5,59,6,69-tetrachloro-1,19,3,39-tetraethylbenzimidazol-carbocyanine, Sigma). The cytofluorimetric analysis was performed collecting green and orange fluorescence for JC-1 staining in 10,000 events for each sample.
## Evaluation of lipid peroxidation
Lipid peroxidation was determined by measuring the thiobarbituric acid (TBA, Inalco, Italy) reactive substances (TBAreactants). Plasma treated HSCs and ISEMFs were incubated for 5 min or 6 hrs. At the end of the incubation, 20% w/vol cold trichloroacetic acid was added to cells and centrifuged. Supernatants were incubated with 0.67% solution of TBA at 100uC for 10 min. The absorbance was recorded at 532 nm. Values were plotted on a standard curve obtained by serial dilution of malonaldehyde tetrabutylammanium salt (Sigma) and the TBAreactant level was calculated using a molar extinction coefficient of 1.56610 5 M 21 6cm 21 . The results for each sample were normalized to the protein concentration determined by bicinchoninic acid assay (Pierce, Italy).
## Cell death assay
Apoptotic loss of plasma membrane asymmetry or necrotic loss of plasma membrane integrity were assessed at 1, 6, 12, 24, and 48 hrs after plasma treatment by labeling cells with Annexin V-FITC and propidium iodide (PI), according to the manufacturer's instructions (Annexin V Fluos, Roche Diagnostic, Italy). Samples were incubated in the dark for 15 min and then analyzed using a BD FACS-Calibur flow cytometer. Ten thousands events were acquired for each sample.
# Western blot analysis
Plasma treated HSCs and ISEMFs were cultured for 4 hrs. The cells were then washed with ice-cold PBS and subjected to total protein extraction in no-denaturing RIPA buffer, as described elsewhere [bib_ref] Exposure to bacterial cell wall products triggers an inflammatory phenotype in hepatic..., Brun [/bib_ref]. Forty mg of proteins was separated for each sample in 7% w/vol SDS-PAGE and finally transferred to PVDF membrane (BioRad, Italy). Membranes were probed with antirabbit peroxisome proliferator activated receptor (PPAR)-c antibody at 4uC for 16 hrs and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody. Anti-b-actin antibody (Sigma) was used as the loading control. Immunecomplexes were visualized using enhanced chemiluminescence (ECL, Millipore). Images were captured using a Hyper film MP (Sigma).
## Cytokine elisa
HSCs and ISEMFs were incubated for 30 min with PPAR-c receptor antagonist T0070907 (50 mM, Tocris, UK) or with vehicle alone. Interleukin (IL)-1 and IL-6, tumour necrosis factor (TNF)-a and transforming growth factor (TGF)-b1 were assessed in the conditioned medium 24 hrs after plasma treatment using commercially available ELISA kits (e-Bioscience, Prodotti Gianni, Italy). Optical densities were measured at 450 nm using a micro plate reader (Sunrise, Tecan; Switzerland). The sensitivity of the assays ranged between 1 and 15 pg/ml.
## Proliferation assay
Cells (3610 5 /ml) were incubated at 37uC for 10 min in prewarmed PBS containing 0.1% vol/vol BSA (Sigma) and 25 mM carboxyfluorescein diacetate succinimidyl ester (CFSE, Molecular Probe, Invitrogen). Staining was quenched by adding 5 volumes of ice-cold culture media. Sixteen hrs later the cells were washed, counted using Trypan blue, seeded at 8610 4 cells/ml, and treated with NAC, as described elsewhere, or with 5 mg/ml cytochalasin B (Sigma). Cultures were then exposed to plasma and subsequently incubated in fresh culture medium at 37uC for 72 hrs. Cell proliferation was evaluated by the partitioning of fluorescent dye between daughter cells using BD FACS-Calibur flow cytometer.
## Migration assay
The cells were labelled for 10 min at 37uC with 5 mM 6carboxyfluorescein diacetate (6-CFDA, Molecular Probe, Invitrogen) cell-permeable esterase substrate and exposed to plasma. The cells were then seeded (8610 4 cells/ml) in DMEM supplemented with 0.2% vol/vol FBS into trans-well inserts (8 mm pore size, Corning, the Netherlands), held in 12-well tissue culture plates containing complete culture medium and cultured for 72 hrs. The bottom side of the trans-well inserts was washed twice and the cells were harvested using cell scrapers. The cell membrane was dissolved in PBS containing 0.5% vol/vol Triton X-100. The samples were then centrifuged at 4uC 13,000 rpm for 10 min. Fluorescence was detected in the supernatant at excitation and emission wavelengths of 485 and 530 nm, respectively (Hitachi F2000 Fluorescence Spectrophotometer, Japan).
## Wound healing assay
Plasma treated cells were seeded onto glass coverslips and cultured at 37uC for 24 hrs. The cell monolayer was then wounded with a plastic tip. Seventy-two hrs later, the cells were rinsed twice in PBS, fixed in paraformaldehyde (PFA) 4% w/vol for 10 min, and routinely stained with haematoxylin and eosin. Finally, the slides were examined using a light transmission . Schematic representation of helium generated plasma source. The plasma source used in the present study consisted of two coaxial tubes, the outer one made out of copper and the inner one made out of insulating material. Two parallel grids 1 mm away from one another closed the tubes. The electric field was formed in the space between the two grids by applying a RF voltage difference supplied by a radiofrequency (RF) generator coupled to the source by a matching network. doi:10.1371/journal.pone.0104397.g001 microscope connected to a camera to capture the images (DMLB Leica, Wetzlar, Germany).
## Statistical analyses
Experiments were performed using HSCs freshly isolated from the liver specimens of 3 different subjects and ISEMFs freshly prepared from the colonic biopsies collected from 3 different subjects. Experiments were carried out two or three times in duplicate or triplicate. The graphics express data as mean 6 standard error (SEM). The unpaired Student's t-test was used to compare the data of two groups and one-way ANOVA test followed by the Newman-Keuls post-hoc test was used to compare the data of three or more groups. Statistical analysis was performed using GraphPad Prism 3.03 (San Diego, California, USA).
# Results
The gap between the plasma source tip and the sample affected intracellular ROS generation Several studies have reported that plasma effects are primarily due to ROS generation [bib_ref] Disinfection of ocular cells and tissues by atmospheric-pressure cold plasma, Brun [/bib_ref] [bib_ref] The emerging role of reactive oxygen and nitrogen species in redox biology..., Graves [/bib_ref]. A number of parameters such as the gas mixture, the plasma power applied, the length of exposure, the composition of the medium embedding the sample, and the distance from the plasma source tip actually affects ROS generation and in the end plasma performance [bib_ref] Nonthermal dielectric-barrier discharge plasma-induced inactivation involves oxidative DNA damage and membrane lipid..., Joshi [/bib_ref] [bib_ref] ROS implication in a new antitumor strategy based on non-thermal plasma, Vandamme [/bib_ref]. In the attempt to evaluate the experimental parameters influencing intracellular ROS generation in this study fibroblast-like primary cells were exposed to plasma for 1, 2, 5, and 10 min. As outlined in , 2 min of plasma treatment significantly increased ROS levels in both HSCs and ISEMFs. Plasma exposure for 1 min did not affect the generation of intracellular ROS, but at longer treatment times (e.g. 5 and 10 min) there was no further increase in intracellular ROS levels with respect to that following 2 min of treatment. In addition, the distance between the plasma source tip and the sample had an important effect on ROS generation. In order to assess this variable fibroblast-like primary cells were exposed for 2 min to plasma at different distances from the source tip. As demonstrated in , intracellular ROS levels were strongly influenced in both HSCs and ISEMFs by the distance from the plasma source tip and they peaked when the gap was fixed at 1.5 mm from the surface of the medium soaking the samples. This was the minimal distance we registered to avoid contact with the medium surface. Subsequent experiments were thus performed at this distance for 2 min of plasma exposure. Under these conditions intracellular ROS formation was not evident when the cells were exposed to the electric field without plasma, thus ruling out the role of the electric field alone in ROS formation (data not shown).
As outlined elsewhere with regard to other primary cell populations [bib_ref] Disinfection of ocular cells and tissues by atmospheric-pressure cold plasma, Brun [/bib_ref] , plasma-generated ROS increased 5 min after plasma treatment in both HSCs and ISEMFs . Plasmagenerated ROS were characterized by short half-lives with respect to ROS induced in cells exposed to H 2 O 2 . In HSCs, ROS were extinguished within 6 hrs following plasma treatment while significant levels of enhanced H 2 DCFDA positive cells were still detectable even 24 hrs after H 2 O 2 challenge. Similar results were obtained in ISEMFs .
## Plasma exposure increased ros level in cytoplasmic compartment
In the attempt to investigate mitochondrial involvement in intracellular ROS generation fibroblast-like primary cells were loaded with JC-1, a dye switching from orange fluorescent aggregates under high mitochondrial potential to green fluorescent monomeric forms after depolarization of the mitochondrial membrane [bib_ref] JC-1, but not DiOC6(3) or rhodamine 123, is a reliable fluorescent probe..., Salvioli [/bib_ref]. As outlined in , incubation with H 2 O 2 considerably increased the percentage of HSCs and ISEMFs with collapsed membrane potential at almost all the time points tested. On the contrary, plasma treatment did not significantly affect mitochondrial depolarization in the fibroblastlike primary cells, and this suggests that mitochondria are not involved in plasma-induced ROS generation.
Plasma-induced intracellular ROS levels were, however, dampened by cellular pre-treatment with different anti-oxidant agents. Thus, as shown in , plasma-elicited ROS generation was significantly blocked by NAC, GSH, and atocopherol (P,0.05 vs plasma-treated cells), all ROS scavengers largely used in clinical therapy, that are able to increase the cytoplasmic antioxidant pool [bib_ref] N-acetylcysteine (NAC) and glutathione (GSH): antioxidant and chemopreventive properties, with special reference..., Van Zandwijk [/bib_ref]. Moreover, ROS formation was mainly prevented in HSCs by catalase indicating that hydrogen peroxide is a critical plasma by-product. Taken together, these data suggest that ROS were generated in the cytoplasm of plasmaexposed primary cells via oxidation of sulphide groups and/or lipids. Indeed, TBA-reactive substances, a marker of lipid peroxidation, increased in the cytoplasmic compartment of both HSCs and ISEMFs 5 min after plasma treatment but disappeared following 6 hrs of incubation , a time course matching the kinetics of ROS levels .
Plasma treatment, however, did not result in cellular death. Thus even after a long incubation period (48 hrs) following plasma treatment, the percentages of Annexin V positive cells as well as Annexin V and PI double positive cells did not significantly increase in HSCs and ISEMFs with respect to those in non-treated cells. On the contrary H 2 O 2 exposure significantly augmented the percentage of late apoptotic/necrotic cells starting 6 hrs after the stimulation [fig_ref] Table 1: Evaluation of cell death [/fig_ref].
## Plasma treatment modulated ppar-c expression and cytokines production
Intracellular ROS act as transducing molecules in mammalian cells and engage different signaling pathways [bib_ref] Low-level laser therapy activates NF-kB via generation of reactive oxygen species in..., Chen [/bib_ref] [bib_ref] Lactobacillus crispatus M247-derived H2O2 acts as a signal transducing molecule activating peroxisome..., Voltan [/bib_ref]. PPAR-c, in particular, is a transcription factor mainly involved in lipid metabolism and regulation of inflammation. Thus, PPAR-c suppresses uncontrolled activation of HSCs and prevents inflammatory damage during colitis [bib_ref] Activation of peroxisome proliferator-activated receptor-gamma contributes to the inhibitory effects of curcumin..., Xu [/bib_ref]. As outlined in [fig_ref] Figure 4: Intracellular signalling of plasma-induced ROS [/fig_ref] , plasma-generated ROS increased the expression of PPAR-c in fibroblast-like primary cells 4 hrs after treatment, but pretreatment of cells with the antioxidant agent NAC prevented the plasma-induced increase in PPAR-c expression.
Plasma-induced activation of the PPAR-c signaling pathway resulted in an increased production and secretion of IL-6, a cytokine involved in tissue regeneration [bib_ref] Protective effects of interleukin-6 in lipopolysaccharide (LPS)-induced experimental endotoxemia are linked to..., Nandi [/bib_ref]. Plasma-treated cells were cultured for 24 hrs at 37uC, and IL-6 expression in conditioned medium was assessed by ELISA. Plasma exposure increased IL-6 levels by 28% in the conditioned medium of HSCs and by 46% in that of ISEMFs. Cell pre-treatment with the PPAR-c receptor antagonist T0070907 blunted the increase in IL-6 levels [fig_ref] Figure 4: Intracellular signalling of plasma-induced ROS [/fig_ref]. Plasma treatment did not increase the production of IL-1b [fig_ref] Figure 4: Intracellular signalling of plasma-induced ROS [/fig_ref] , TNF-a, or TGF-b (data not shown) in HSCs and ISEMFs.
## Plasma exposure promoted cell proliferation and migration
Cell proliferation was assessed evaluating the partitioning between daughter cells of CFSE probe-related fluorescence in cells cultured for 72 hrs, a condition established in our preliminary experiments. According to FACS analysis [fig_ref] Figure 5: Proliferation and migration in plasma treated fibroblast-like cells [/fig_ref] , the fluorescence partitioning occurred in 15.9% of non-treated HSCs while plasma exposure stimulated proliferation in 49.8% of the cells. Cellular proliferation occurred in only 1.97% of HSCs pretreated with cytochalasin B, an inhibitor of cell division [fig_ref] Figure 5: Proliferation and migration in plasma treated fibroblast-like cells [/fig_ref]. Similar results were obtained in the ISEMFs (data not shown). The plasma-induced cell proliferation was secondary to intracellular ROS generation since NAC, a ROS scavenger, significantly abolished cellular division (P,0.05 vs plasma-treated cells, [fig_ref] Figure 5: Proliferation and migration in plasma treated fibroblast-like cells [/fig_ref].
Besides increasing cellular proliferation, plasma treatment promoted migration of fibroblast-like primary cells. As outlined in [fig_ref] Figure 5: Proliferation and migration in plasma treated fibroblast-like cells [/fig_ref] , migration of plasma-exposed HSCs and ISEMFs labeled with 6-CFDA probe increased respectively by 2.67 fold and 1.62 fold as compare to non-treated cells. Again, cell migration was abolished by NAC pretreatment, demonstrating that plasma-induced cell migration is mediated by intracellular ROS formation. As a consequence of the increased cell proliferation and migration, plasma-treated ISEMFs also demonstrated marked ability in gap closure assay [fig_ref] Figure 5: Proliferation and migration in plasma treated fibroblast-like cells [/fig_ref].
# Discussion
Wound care and therapy have been drawing growing attention particularly in view of the increase in diseases associated with skin and inner lesions due to aging, diabetes and trauma. It is also known that atherosclerosis and defective nutrition can delay or jeopardize the repair process. The options available today to care for acute or chronic lesions are numerous but at the same time, several factors work against the repair/regeneration process [bib_ref] Overview: acute and chronic wounds, Whitney [/bib_ref]. According to the most up to date findings, normal wound repair after tissue injury follows a closely regulated sequence of events including the activation and proliferation of fibroblastic-like cells. In pathological situations the normal stages are altered and those processes continue inducing excessive accumulation of extracellular matrix [bib_ref] Wound repair and regeneration, Gurtner [/bib_ref] [bib_ref] Role of myofibroblasts during normal tissue repair and excessive scarring: interest of..., Badid [/bib_ref]. In view of these considerations, medical tools able to induce transient activation of fibroblast-like cells could be useful in promoting healing of lesions without inflammation and scarring [bib_ref] Applied Plasma Medicine, Fridman [/bib_ref]. In the present study we have demonstrated that nonthermal atmospheric pressure plasma ionizing helium gas mixed with air generates active chemical species that tune the activation of two fibroblast-like cell populations (human HSCs and ISEMFs) by inducing intracellular ROS.
For decades ROS have been considered accidental byproducts of oxygen metabolism having detrimental effects on cell survival. More recent investigations have however uncovered the dual nature of ROS and their role in sustaining normal biological functions [bib_ref] Free radicals and antioxidants in normal physiological functions and human disease, Valko [/bib_ref] [bib_ref] Cell signaling. H2O2, a necessary evil for cell signaling, Rhee [/bib_ref]. On the one hand, higher than normal ROS levels cause irreversible damage to cellular organelles, membrane, proteins and DNA; they have also been implicated in ageing, cancer, and neurodegenerative diseases [bib_ref] The mitochondrial free radical theory of aging: a critical view, Sanz [/bib_ref] [bib_ref] Toxicological and pharmacological concerns on oxidative stress and related diseases, Saeidnia [/bib_ref]. On the other, cells have evolved mechanisms such as antioxidant enzymes to modulate intracellular ROS levels [bib_ref] Cell responses to oxidative stressors, Cataldi [/bib_ref]. Thus, at low/moderate concentrations ROS activate the intracellular signaling pathways resulting in production of soluble factors involved in cell growth and proliferation [bib_ref] Cell signaling. H2O2, a necessary evil for cell signaling, Rhee [/bib_ref]. ROS generated in human HSCs and . Plasma-induced intracellular ROS levels. Primary cultured human hepatic stellate cells (HSCs) and intestinal subepithelial myofibroblasts (ISEMFs) were loaded with H 2 DCFDA and exposed for 1, 2, 5, or 10 min to helium-generated plasma. The cells were cultured for 5 min under optimal conditions and then evaluated for intracellular ROS generation by FACS analysis. ROS formation was expressed as the percentage of fluorescence relative to intracellular ROS (ROS fluorescence %, panel A). Intracellular ROS generation was evaluated in fibroblast-like primary cells exposed for 2 min at different distances from the plasma source tip (panel B). The gap between the plasma source tip and the sample was fixed at 1.5 mm, and intracellular ROS generation was evaluated at different times of incubation after 2 minutes of plasma treatment or following exposure to 20 mM H 2 O 2 (panel C). During FACS analysis 10,000 events were collected. Data are reported as mean6SE of the percentage of fluorescence registered in nine independent experiments, each performed in duplicate or in triplicate. * denotes P,0.05 vs respective not treated cells (CTR). doi:10.1371/journal.pone.0104397.g002
ISEMFs by plasma exposure were found here to be characterized by lower concentrations and shorter half-lives with respect to H 2 O 2 induced ROS . The transient increase in ROS levels spared cells early markers of apoptosis such as the decrease in the mitochondrial membrane potential and the externalization of phosphatidylserine [fig_ref] Table 1: Evaluation of cell death [/fig_ref] , an important . Sources of ROS in plasma-treated fibroblast-like primary cells. HSCs and ISEMFs were loaded with JC-1 and exposed to plasma or to 20 mM H 2 O 2 . Cells were cultured for 1, 6, 24 and 48 hrs under optimal conditions and mitochondrial dysfunction was evaluated by FACS analysis in 10,000 events (panel A). H 2 DCFDA-loaded cells were treated for 15 min with 5 mM N-acetyl L-cysteine (NAC), 20 mM reduced glutathione (GSH), 50 mM a-tocopherol, 1000 U/ml catalase and exposed for 2 min to plasma. After 5 min, the percentage of fluorescence relative to intracellular ROS was evaluated by FACS analysis in 10,000 events (panel B). Cells were treated with plasma for 2 min and incubated under standard conditions for 5 min or 6 hrs. Thiobarbituric acid (TBA)-reactants were evaluated in cell lysates and normalized to protein contents (panel C). Data are reported as mean6SE of results collected in nine independent experiments, each performed in duplicate or triplicate. u denotes P,0.02 vs non treated cells. 1 denotes P,0.05 vs non treated cells (CTR). * denotes P,0.05 vs plasma-treated cells. doi:10.1371/journal.pone.0104397.g003 Plasma generated ROS were found to increase PPARc expression [fig_ref] Figure 4: Intracellular signalling of plasma-induced ROS [/fig_ref] , the transcription factor belonging to the nuclear hormone receptor superfamily. In recent years the pleiotropic effects of PPAR-c agonists (e.g. regulation of energy metabolism, suppression of inflammatory activity, control of tissue remodeling) have become evident. PPAR-c, nevertheless, engages different signaling pathways in a cell specific fashion mainly through the net balance of the soluble factors produced [bib_ref] Lactobacillus crispatus M247-derived H2O2 acts as a signal transducing molecule activating peroxisome..., Voltan [/bib_ref] [bib_ref] Activation of peroxisome proliferator-activated receptor-gamma contributes to the inhibitory effects of curcumin..., Xu [/bib_ref] [bib_ref] PPAR-gamma agonists inhibit production of monocyte inflammatory cytokines, Jiang [/bib_ref]. In cultured human HSCs and ISEMFs, plasma triggered the production and secretion of IL-6, cell migration, and proliferation and prompted the closure of the gap, as was assessed by the in vitro wound healing model [fig_ref] Figure 4: Intracellular signalling of plasma-induced ROS [/fig_ref]. Cellular migration is crucial during the wound healing process and is regulated by several intra-and extra-cellular mechanisms. Actin polymerization, for example, produces force that alters the cytoskeletal structures and leads to cell adhesion and spreading in the immediate vicinity of the wound [bib_ref] Actin-based cell motility and cell locomotion, Mitchison [/bib_ref]. The signals governing the uniform directionality of cell migration toward the repairing area are, however, not entirely understood [bib_ref] Wound repair and regeneration, Gurtner [/bib_ref]. Nishimura and colleagues reported that human keratinocytes move to the negative pole of the electric fields generated near wounds in mammalian skin [bib_ref] Human keratinocytes migrate to the negative pole in direct current electric fields..., Nishimura [/bib_ref]. The electrically directed movement, known as galvanotaxis, is not, however, shared by all cells involved in the wound healing process [bib_ref] Melanocytes do not migrate directionally in physiological DC electric fields, Grahn [/bib_ref]. Findings from the present study demonstrated that migration and proliferation in the plasma-exposed HSCs and ISEMFs are ROS dependent since biological effects were significantly reduced by pre-treatment of cells with the NAC antioxidant agent [fig_ref] Figure 5: Proliferation and migration in plasma treated fibroblast-like cells [/fig_ref] and C). Indeed, a growing body of studies published by Lindequist U. and co-workers has clearly demonstrated that in the non-tumorigenic human keratinocyte cell line HaCaT plasma-generated ROS influences the expression of the surface pattern receptors (e.g. integrins) mainly involved in cellular protrusion [bib_ref] Differential influence of components resulting from atmospheric-pressure plasma on integrin expression of..., Haertel [/bib_ref] [bib_ref] Non-thermal atmospheric-pressure plasma can influence cell adhesion molecules on HaCaT-keratinocytes, Haertel [/bib_ref] [bib_ref] Influence of non-thermal atmospheric pressure plasma on cellular structures and processes in..., Blackert [/bib_ref] [bib_ref] Atmospheric pressure plasma jet treatment evokes transient oxidative stress in HaCaT keratinocytes..., Wende [/bib_ref]. It is well known, nevertheless, that plasma components (such as radiations, ionized nitrogen or ROS), plasma power, the distance from the sample and exposure time affect cells differently. The numerous parameters influencing the performance of various plasmas could explain the differences in cell death and cell migration that have been outlined in other works [bib_ref] Differential influence of components resulting from atmospheric-pressure plasma on integrin expression of..., Haertel [/bib_ref] [bib_ref] Plasmacontrolled cell migration: localization of cold plasma-cell interaction region, Volotskova [/bib_ref]. As clearly described in our own study, even the levels of intracellular ROS are important in tuning the cellular effects and [fig_ref] Table 1: Evaluation of cell death [/fig_ref] , plasma vs H 2 O 2 treatment). Volotskova O. and colleagues, moreover, reported that following 100 s of treatment the reduction in fibroblast migration was affected by the distance from the treated area. Cells had the slowest migration rate in the plasma treated zone while those outside the treated area (5-8 mm away) migrated at the same rates as the untreated cells [bib_ref] Plasmacontrolled cell migration: localization of cold plasma-cell interaction region, Volotskova [/bib_ref]. Even if the authors did not investigate intracellular signaling, it seems that their plasma jet did not generate reactive diffusible ROS.
Mitochondria, peroxisome, cytoplasmic proteins and lipid layers constitute the main sources of ROS in eukaryotic cells. Our findings demonstrated that 2 min of treatment with helium generated plasma does not alter the mitochondrial membrane potential in fibroblasts thus excluding these organelles as source of ROS . Administration of cytoplasmic free radical scavengers, on the other hand, significantly dampened the plasmainduced generation of ROS suggesting that cytoplasmic proteins and membrane lipids are the eligible substrate for ROS formation after plasma exposure. Indeed, plasma treatment induced early transient lipid peroxidation in the cytoplasmic compartment . The rapid drop in plasma-induced ROS generation prompted us to hypothesize that just as other human primary cells [bib_ref] Disinfection of ocular cells and tissues by atmospheric-pressure cold plasma, Brun [/bib_ref] , HSCs and ISEMFs are endowed with an efficient armamentarium of scavenger enzymes to contain the oxidative burst and to avoid mitochondrial involvement thus preserving ATP production. ROS kinetics elicited in primary cells by plasma treatment significantly differed from those described in Representative Western blot analysis of PPAR-c in protein extracts from HSCs and ISEMFs treated with plasma and incubated for 4 hrs. As indicated, cells were pre-treated with NAC. Similar results were obtained in three independent experiments. b-actin was used as a loading control (panel A). Cells were treated for 30 min with 50 mM PPAR-c receptor antagonist T0070907, exposed to plasma, and then incubated under standard conditions for 24 hrs. IL-6 (panel B) and IL-1b (panel C) were assessed in the conditioned medium by ELISA. Data are reported as mean6SE of results collected in nine independent experiments, each performed in triplicate. * denotes P,0.05 vs non treated cells. doi:10.1371/journal.pone.0104397.g004 tumor cells where hypoxia and nutrient deprivation usually result in mitochondrial dysfunctions boosting ROS production and accounting for an increased rate of death of tumor cells in response to oxidative stress (our unpublished data) [bib_ref] Lowtemperature plasma-induced antiproliferative effects on multi-cellular tumor spheroids, Plewa [/bib_ref] [bib_ref] The causes of cancer revisited: ''mitochondrial malignancy'' and ROSinduced oncogenic transformation -why..., Suralph [/bib_ref].
Our results have clearly demonstrated that helium generated plasma treatment induces proliferation and migration of human fibroblast-like primary cells mainly through intracellular ROS formation. Since the threshold of ROS is crucial in cellular signaling and their levels could be modulated in the intracellular compartment by non-thermal atmospheric pressure plasma, it represents a promising tool in the control of fibroblast activation. As outlined in , differences of only fractions of a millimeter in the distance between the plasma needle and the cellular medium affect ROS generation. Moreover, as recently reported, the composition of the medium embedding the samples greatly influences ROS generation in plasma treated cells [bib_ref] Atmospheric pressure plasma jet treatment evokes transient oxidative stress in HaCaT keratinocytes..., Wende [/bib_ref]. Taken together, these data demonstrate that the effects of plasma can be modulated depending on the nature of the sample and the aim of the treatment. At the same, however, it is by now unquestionable that in vivo plasma treatment requires availability of accurate standardized devices as well as in depth knowledge about the biological significance of ROS formation in different cell populations. While more studies are required to investigate DNA damage induced by low transient levels of ROS in primary cells, additional clinical trials investigating the tolerability of plasma treatments [bib_ref] Plasma medicine: an introductory review, Kong [/bib_ref] and further developments in the technology of atmospheric cold plasma generated using small needles will hopefully pave the way for endoscopic applications in the event of liver and gut lesions.
[fig] 9: Windows Multiple Document Interface for Flow Cytometry) program. In a different set of experiments, the cells were treated for 15 minutes at 37uC with 5 mM N-acetyl L-cysteine (NAC), 20 mM glutathione (GSH), 50 mM a-tocopherol, 1000 U/ml catalase or 20 mM H 2 O 2 (all provided by Sigma), all conditions previously established in our laboratories. [/fig]
[fig] Figure 4: Intracellular signalling of plasma-induced ROS. [/fig]
[fig] Figure 5: Proliferation and migration in plasma treated fibroblast-like cells. HSCs were labelled with 25 mM CFSE and incubated with NAC or 5 mg/ml cytochalasin B. Cells were then treated with plasma and incubated for 72 hrs. Panel A outlines representative FACS analysis of cellular proliferation assessed by evaluating the partition of CFSE. Similar results were obtained in nine independent experiments, each performed in triplicate. Panel B reports the percentage of proliferating fluorescent cells. Panel C reports the fluorescence relative to 6-CFDA in cells migrating to the bottom side of trans-well inserts (a.u. means arbitrary unit). Data are reported as mean6SE of results collected in nine independent experiments, each performed in triplicate. u denotes P,0.02 vs non treated cells. * denotes P,0.05 vs not treated cells. Plasma treated ISEMFs were seeded onto glass coverslips. The cell monolayer was wounded using a plastic tip and 72 hrs later the cells were stained with haematoxylin and eosin. Images were observed and captured using a light transmission microscope connected to a camera (DMLB Leica, Panel D). Similar results were obtained in three independent experiments. doi:10.1371/journal.pone.0104397.g005 [/fig]
[table] Table 1: Evaluation of cell death. [/table]
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Spatial Distribution of Stony Desertification and Key Influencing Factors on Different Sampling Scales in Small Karst Watersheds
Karst areas are typical ecologically fragile areas, and stony desertification has become the most serious ecological and economic problems in these areas worldwide as well as a source of disasters and poverty. A reasonable sampling scale is of great importance for research on soil science in karst areas. In this paper, the spatial distribution of stony desertification characteristics and its influencing factors in karst areas are studied at different sampling scales using a grid sampling method based on geographic information system (GIS) technology and geo-statistics. The rock exposure obtained through sampling over a 150 m × 150 m grid in the Houzhai River Basin was utilized as the original data, and five grid scales (300 m × 300 m, 450 m × 450 m, 600 m × 600 m, 750 m × 750 m, and 900 m × 900 m) were used as the subsample sets. The results show that the rock exposure does not vary substantially from one sampling scale to another, while the average values of the five subsamples all fluctuate around the average value of the entire set. As the sampling scale increases, the maximum value and the average value of the rock exposure gradually decrease, and there is a gradual increase in the coefficient of variability. At the scale of 150 m × 150 m, the areas of minor stony desertification, medium stony desertification, and major stony desertification in the Houzhai River Basin are 7.81 km 2 , 4.50 km 2 , and 1.87 km 2 , respectively. The spatial variability of stony desertification at small scales is influenced by many factors, and the variability at medium scales is jointly influenced by gradient, rock content, and rock exposure. At large scales, the spatial variability of stony desertification is mainly influenced by soil thickness and rock content.
# Introduction
Soil is a continuum with uneven changes, and soil properties present obvious spatial variability [bib_ref] Average variograms to guide soil sampling, Kerry [/bib_ref]. The foundation of soil science research is to obtain detailed and accurate spatial distribution information on soil properties [bib_ref] Effects of land cover on soil organic carbon stock in a karst..., Chen [/bib_ref]. The sampling scale has a decisive influence on the acquisition accuracy and quantitative expression of soil properties and spatial variability information [bib_ref] Comparing sampling needs for variograms of soil properties computed by the method..., Kerry [/bib_ref]. Theoretically, the narrower the sampling scale, the lower the prediction error of an interpolation. If sampling is oversized, it is difficult to guarantee interpolation accuracy [bib_ref] Optimum size in grid soil sampling for variable rate application in site-specific..., Marcos [/bib_ref]. However, excessively high sampling density will require more manpower, material resources, financial resources and long work cycles [bib_ref] Effect of soil sampling density on detected spatial variability of soil organic..., Yu [/bib_ref]. How to determine a reasonable sampling density during regional research on soil science is a key and difficult point of the present research on soil science [bib_ref] Sample adequately to estimate variograms of soil properties, Webster [/bib_ref]. Different sampling scales lead to different characteristics, including different influencing factors and different evolution mechanisms and processes [bib_ref] Variability of variograms and spatial estimates due to soil sampling: A case..., Gascuel-Odoux [/bib_ref]. Therefore, the key influencing factors can be reliably revealed only if the sampling scale is in line with the intrinsic scale of the phenomenon to be studied. In karst areas, the earth surface is greatly uneven, the landscape is fragmented, and the key influencing factors vary with sampling scale.
The concept of stony desertification was first proposed in the early 1980s, and later, it was defined as a term representing the process of the transition from vegetation-covered and soil-covered karst areas to karst landscapes covered by bare rocks [bib_ref] Water-storage capacity controls energy partitioning and water use in karst 470 ecosystems..., Heilman [/bib_ref]. Karst areas in China are typical ecologically fragile areas, and the problem of stony desertification has become the most serious ecological and economic problem as well as a source of disasters and poverty in the area [bib_ref] The Evolution of a Karst Rocky Desertification Land Ecosystem and Its Driving..., Li [/bib_ref]. According to the experience in treating typical stony desertification areas, the treatment requires guidance on the driving mechanisms and theories at different spatial scales [bib_ref] The German Agricultural Soil Inventory: Sampling design for a representative assessment of..., Bach [/bib_ref]. For example, a great number of studies regarding soil organic carbon at different scales and in different landforms have been conducted over the past several decades [bib_ref] Spatial variability of soil organic carbon and representative soil sampling method in..., Wang [/bib_ref]. However, the results of these studies differ significantly from each other. For instance, the reported global soil organic carbon storage ranged from 504 Pg to 3000 Pg with a mean value of 1460.5 Pg. One of the main reasons for this variation is the high spatial variability in the organic carbon content of soils, which is caused by various landforms and complicated changing geological conditions [bib_ref] Organic carbon density and storage in soils of China and spatial analysis, Xie [/bib_ref]. In particular, it is necessary to determine the positive and negative impacts of natural and human effects on the progress of stony desertification as well as their respective contribution rates [bib_ref] Dissolved organic carbon and its carbon isotope compositions in hill slope soils..., Liu [/bib_ref]. Therefore, the distribution characteristics of stony desertification and their relations with environmental factors on different sampling scales are of great importance for the understanding of the progress of a karst ecosystem and have received widespread attention [bib_ref] Soil organic carbon in the Sanjiang Plain of China: Storage, distribution and..., Mao [/bib_ref]. At present, the majority of studies on the spatial distribution of stony desertification are limited to single factors and the spatial correlation [bib_ref] Limestone Karsts of Southeast Asia: Imperiled Arks of Biodiversity, Reuben [/bib_ref]. There are very few comprehensive and systematic quantitative analyses of the impacts of various factors on the spatial distribution of stony desertification, and there are even fewer studies on problems about differences in scale. In this paper, GIS and geo-statistics are combined to reveal patterns of the spatial distribution characteristics of stony desertification at different sampling scales. Multiple stepwise regression and Pearson correlation analysis are used to explore the key influencing factors of stony desertification characteristics on non-sampling scales. These methods are aimed at revealing the determinants of the spatial distribution of stony desertification and the differences among different scales, and the results provide references for the treatment of stony desertification in karst areas.
# Materials and methods
## Study area
The study region
## Data source
Sampling plots were designed with a grid-based sampling method, and there was a total of 2755 sampling grids (150 m × 150 m) consisting of 22,057 soil samples. The sampling depth is 100 cm. The sampling sites were defined as the center of each sampling grid [fig_ref] Figure 1: The location of Houzhai River basin and the distribution of sample sites [/fig_ref]. The soil samples were air dried, ground and prepared for analysis as required by the laboratory. Rock exposures were measured by linear interception using tape in the field. Rock exposure was assessed by the average percentage of rock occupied in 10 m distance, and the texts were repeated for four times in east, west, north and south direction, respectively.
## Classification of different grid scales
The grid scale determines the density and sampling point data. To study the impacts of different sampling scales on the revelation of spatial variability of soil organic carbon, ArcGIS software is adopted to extract cells from the 2755 grids at 150 m × 150 m to obtain 802 grids of 300 m × 300 m; then, these grids were classified into six grid scales: 150 m × 150 m, 300 m × 300 m, 450 m × 450 m, 600 m × 600 m, 750 m × 750 m and 900 m × 900 m [fig_ref] Figure 2: Plots of soil sample distribution under different sampling scales [/fig_ref]. At the same time, to ensure the reliability of the conclusions and reduce the unreliability resulting from repeated sampling, subsets of five samples were selected without replacement.
## Calculations and statistical analysis
Classical statistical methods can be used to describe some overall characteristics of the coverage of rock exposures (CRE), but they are unable to characterize its spatial variability. Therefore, this study used a geostatistical method to quantify the constitutive properties and randomness of the CRE to analyze the patterns of spatial variation in the CRE more accurately [bib_ref] Spatial variability of surface sediment basis on geostatistical analysis in the littoral..., Zhang [/bib_ref].
A semivariance function (h) was used to describe the spatial heterogeneity of the soil properties. The semivariance function was used to obtain the variation in the semivariance function value with an increase in the distance of the sample; the scatter plots were fitted with a Gaussian model and other theoretical models. When the soil properties met a two-order stationary assumption and the intrinsic hypothesis and when the sample size was large enough, the semivariance theory variation function (h) formula was used. The semivariance (r(h)) is as follows [bib_ref] Spatial Dependence of Soil Enzyme Activities along a Slope, Bergstrom [/bib_ref] :
[formula] r(h) = 1 2N(h) N(h) ∑ i=1 [Z(xi) − Z(xi + h)] 2 ,(1) [/formula]
where Z is the measured soil property, x is the sample location, and N(h) is the number of pairs of locations separated by a lag distance h. The semivariogram expresses the relationship between the semivariance and the lag distance (h). The semivariogram typically increases from a value at h = 0 (identified as the nugget) to a maximum value (identified as the sill), which is created using ArcGIS software. The rock exposure of the spatial distribution pattern was determined using a kriging interpolation method with a spatial interpolation grid. Statistical analyses were performed using SPSS18.0 (SPSS Inc., IBM Corporation, Chicago, IL, USA) and Excel2007 (Office 2007, Redmond, WA, USA). A semivariogram model, fitted with GS 9.0+ software (Gamma Design Software, GS + 9.0, LLC Plainwell, MI, USA), was used for ordinary kriging interpolation in ArcGIS 9.3 software (ESRI, ArcGIS 9.3, Redlands, CA, USA), rendering a rock exposure spatial distribution map. [fig_ref] Table 1: Description and statistics of rock exposures under different sampling scales [/fig_ref] provides the statistics on the CRE at different sampling scales. The statistics reveal a wide gap between the maximum and minimum CRE in the Houzhai River basin, which are 95.00% and 0.00%, respectively. The CRE varied slightly with the sampling scale. The 150 m × 150 m scale has the highest average CRE at 15.94%, while the 900 m × 900 m scale had the lowest average CRE at 9.89%. The former is 1.62 times greater than the latter. As the sampling scale increases, the maximum and average values of CRE gradually decrease while the coefficient of variation increases. The coefficient of variation (CV) is a measure of dispersion of the distribution of a random variable, i.e., the extent of spatial variability in an attribute indicator. Normally, CV values no greater than 10% indicate low variability, CV values between 10% and 100% indicate moderate variability, and CV values greater than 100% signify high variability. The CV in the CRE across the Houzhai River basin ranges from 139.84% to 197.67% [fig_ref] Table 1: Description and statistics of rock exposures under different sampling scales [/fig_ref] , suggesting high CRE variability at various sampling scales. This result indicates that the coverage of rock exposures exhibits significant spatial variation within the study area. The average CRE values for the five subsamples fluctuate around the average CRE for each sample set, indicating that the statistical characteristics of the subsamples are consistent with those of the sample and, thus, are representative despite the small number of sampling points in each sample. The CV in CRE first increases and then decreases with increasing sampling scale. Moreover, the CRE is normally distributed in each sample set and all subsamples in terms of both skewness and kurtosis.
# Results and analysis
## Descriptive statistics for the coverage of rock exposures at different sampling scales
## Semivariograms describing the coverage of rock exposures at different sampling scales
The CRE data obtained at different sampling scales were fitted with semivariograms using GS+7.0 Software [fig_ref] Table 2: Semi-variance model of rock exposures and its fitting parameters under different sampling... [/fig_ref]. As seen in [fig_ref] Table 2: Semi-variance model of rock exposures and its fitting parameters under different sampling... [/fig_ref] , the CRE values across the study area follow an exponential distribution at different sampling scales. The 150 m × 150 m sample set was used as the reference, and the semivariograms for the five sample subsets were then compared against the semivariogram for the sample set. It was found that the codomain of CRE decreases steadily with increasing sampling scale. This result is possible because a larger sampling scale is associated with a smaller number of samples and thus lower levels of uniformity in the indicators considered. The nugget effect (C 0 ) is usually used to measure the variation due to experimental error and negative deviation from actual sampling scale, i.e., spatial heterogeneity caused by random factors. [fig_ref] Table 2: Semi-variance model of rock exposures and its fitting parameters under different sampling... [/fig_ref] shows that the C 0 peaks in the 150 m × 150 m sample set and declines with increasing sampling scale. This result indicates that the amount of variation caused by random factors tends to decrease as the sample scale increases, possibly because a decrease in sampling scale will increase the number of random factors involved and the complexity of the causes of variability, and thus, more secondary causes will be neglected. The nugget coefficient of the semivariogram, which is defined as C 0 /C 0 + C, is 0.512 for the 150 m × 150 m sample set and 0.500 for the 300 m × 300 m sample set. The nugget coefficient is smaller than 0.5 at the scale of 450 m × 450 m, and it begins to decrease as the sampling scale further increases. This result suggests a strong spatial dependence of CRE at sampling scales greater than 450 m × 450 m. This result is possible because the spatial dependence caused by structural and random factors at small scales is covered by that at larger scales. Therefore, an increase in sampling scale can strengthen the effects of structural factors and thereby lead to variability in spatial patterns within a certain region.
## Spatial characteristics of rocky desertification in a small catchment area in karst
Based on the spatial characteristics of the coverage of bedrock exposures in the Houzhai River basin and methods of rocky desertification classification provided by previous research, this study classifies the extent of rocky desertification in this area into the following grades: (1) non-karst region (NSD) unaffected by rocky desertification: coverage of bedrock exposures < 20%; (2) potential rocky desertification (PSD): 20% ≤ coverage of bedrock exposures < 30%; (3) light rocky desertification (LSD): 30% ≤ coverage of bedrock exposures < 50%; (4) moderate rocky desertification (MSD): 50% ≤ coverage of bedrock exposures < 70%; and (5) severe rocky desertification (HSD): 70% ≤ coverage of bedrock exposures < 90%. The data from the present study show that at the scale of 150 m × 150 m, slight, moderate, and severe rocky desertification cover areas of 7.81 km 2 , 4.50 km 2 , and 1.87 km 2 , respectively, in the Houzhai River basin.
Rocky desertification in the study area is distributed primarily in the peak-cluster depressions from the northwest to the southeast. As the sampling scale increases, the distribution of severe rocky desertification varies significantly in the southeastern region, while the distribution of moderate rocky desertification varies little (shown in [fig_ref] Figure 3: Spatial distribution of rocky desertification under different sampling scales [/fig_ref]. The northern and central parts of the study area exhibit extensive rocky desertification shrinkage, while the coverage of rocky desertification decreases sporadically in the northern part. The expansion of severe rocky desertification is concentrated in the Yuyangzhai and Dayouzhai villages in the southeast and the Chenqi, Houshan, and Zhaojiatian villages in the north. The trend in the extent of rocky desertification with the number of samples can better characterize the actual evolution of rocky desertification. All variations in the rocky desertification of different grades found in the study area are driven by a combination of natural and human factors. The factors affecting rocky desertification vary with sampling scale. Therefore, determining the different driving factors behind the variations can facilitate more reasonable sampling in research on rocky desertification. NSD is non-karst region; PSD is potential rocky desertification; LSD is slight rocky desertification; MSD is moderate rocky desertification; HSD is severe rocky desertification.
## Factors affecting the characteristics of rocky desertification at different sampling scales
As the grade of rocky desertification depends on the CRE, this study uses the factors affecting the CRE to characterize the factors affecting rocky desertification [fig_ref] Table 3: Correlation matrix of rock exposures and its influencing factors under different sampling... [/fig_ref]. A Pearson correlation analysis shows that at the scale of 150 m × 150 m, the CRE has extremely significant and positive correlations with slope and elevation (p < 0.01), and the correlation coefficients are 0.893 and 0.991, respectively. The correlation with soil thickness is extremely significant and negative (p < 0.01), and the correlation coefficient is −0.913. A significantly positive correlation exists between the CRE and slope position (p < 0.05), and the correlation coefficient is 0.480. The correlations between the CRE and soil bulk density and rock content were not significant (p > 0.05). At the scale of 300 m × 300 m, the CRE has a significantly negative correlation with soil thickness (r = −0.732) (p < 0.05) and significantly positive correlations with elevation (r = 0.512) and rock content (r = 0.610) (p < 0.05). The correlations with slope (r = 0.721) and slope position (r = 0.913) are extremely significant and positive (p < 0.01). There is no obvious correlation between CRE and soil bulk density. At the scale of 450 m × 450 m, the CRE has extremely significant and positive correlations with slope, elevation, rock content, and slope position (p < 0.01, [fig_ref] Figure 1: The location of Houzhai River basin and the distribution of sample sites [/fig_ref]. At the scale of 600 m × 600 m, the CRE shows an extremely significant and positive correlation with rock content (p < 0.01, r = 0.684). The CRE has significant, positive correlations with slope and slope position (p < 0.05), and the correlation coefficients are 0.503 and 0.406, respectively. There is no significant correlation between the CRE and other factors. At the scale of 750 m × 750 m, the CRE is positively and significantly correlated with rock content and slope position (p < 0.05), and the correlation coefficients are 0.780 and 0.741, respectively. At the scale of 900 m × 900 m, the CRE has an extremely significant and negative correlation with soil thickness (p < 0.01, r = −0.637) and a strong positive correlation with slope position (p < 0.05), and the correlation coefficient is 0.501. The regression equations fitted to the CRE data are shown in [fig_ref] Table 4: Optimal fitted equations of factors influencing rock exposures at different sampling scales [/fig_ref]. The values of the coefficient of determination (R 2 ) reveal that the distribution of soil bulk density has the poorest fit at all sampling scales, which indicates a relatively weak correlation between soil bulk density and the CRE. As the scale increases, the distributions of topographic factors (elevation and slope) and rock content improve in the goodness of fit to the CRE. Overall, an increase in the sampling scale improves the goodness of fit of the equations. The data obtained at a large scale (e.g., 900 m × 900 m) can describe the relationships between CRE and the factors more accurately, while the data acquired at a small scale (e.g., 150 m × 150 m) is less able to reflect their relationships due to the influence of complex microtopography. Therefore, decreasing the sampling scale may decrease the goodness of fit between the CRE and various influencing factors.
It is clear from the aforementioned findings that the key driving factors behind the spatial variability in the CRE differ depending on the sampling scale. At small scales (150 m × 150 m, 300 m × 300 m and 450 m × 450 m), the spatial variations in the CRE are affected by a combination of slope, elevation, soil thickness, and the CRE. At a medium scale (600 m × 600 m), the spatial variations in the CRE depend on slope, rock content, and CRE. At large scales (750 m × 750 m and 900 m × 900 m), soil thickness and the CRE are the key factors influencing the variability in the CRE. As the sampling scale increases, the structural features attributed to the concentration of multiple complex factors over short distances are hidden by the factors that affect CRE over longer distances, such as soil thickness and the CRE. This result explains why the topographic factors that have relatively stable and continuous distributions (soil thickness and the CRE) show stronger correlations with the CRE at large scales.
# Discussion
## Relations between sampling scale and stony desertification
In general, there are two kinds of understandings about study scales: one is to conduct studies at multiple scales within a fixed study area by encrypting or broadening the sample number, and the other is to conduct studies at multiple scales by changing the study areas from small to large. The two methods reveal different factors or processes and have different characteristics [bib_ref] Representative sampling size for strip sampling and number of required samples for..., Hu [/bib_ref]. In some studies on the effects of soil attributes at multiple scales the first method has been utilized. However, the majority of researchers have utilized the second method. They study the sampling scale affect by expanding the study area. This method put emphasis on deduction based on results of studies at different area, and tries to reveal the relationship between sampling scale and study area [bib_ref] Spatial variability and optimal sampling strategy of soil respiration, Rodeghiero [/bib_ref] [bib_ref] Variation of soil respiration at three spatial scales: Components within measurements intra-site..., Martin [/bib_ref]. The second method cannot meet the demands of multiscale studies within a fixed area to reflect the global scope. Then, a scale can only reflect the information of local soil characteristics within the sampling range, and it is unable to describe global soil characteristics at different sampling scales [bib_ref] Automation and standardisation of site specific soil sampling, Lütticken [/bib_ref]. However, it is inevitable that changes to scale will lead to fluctuations in variability, resulting in deviations between apparent variations and real variations [bib_ref] The challenge and future of rocky desertification control in karst areas in..., Zhang [/bib_ref]. In particular, the karst areas cannot reflect the factors that influence stony desertification areas very well [bib_ref] The heterogeneity and its influencing factors of soil nutrients in peak-cluster depression..., Zhang [/bib_ref]. In view of this, small watersheds were taken as the objects and the study were carried out in an approximately 75 km 2 area in this study. The first method can more effectively reflect soil information on different scales within the entire basin. With a comprehensive grid sampling method being used in the basin, a comparison was made among different sampling scales that are evenly distributed throughout the basin, which effectively avoids the overgeneralization of the results from studies at small and medium scales.
Karst stony desertification is one of the main types of land desertification [bib_ref] Epilithic organic matter and nutrient contents in three different karst, Wang [/bib_ref]. Karst stony desertification occurs in fragile ecological environments and is driven by extremely unreasonable human activities [bib_ref] Spatial variability of soil organic carbon in relation to environmental factors of..., Wei [/bib_ref] [bib_ref] Comparative studies of the distribution characteristics of rocky desertification and land use/land..., Ying [/bib_ref]. The degradation of land productivity is the essence and the appearance of similar desert landscapes is the symbol of stony desertification. The interference of unreasonable human activities exacerbates the evolution and progress of landscape fragmentation in karst landscapes that are characterized by stony desertification. Therefore, accurately reflecting the spatial distribution patterns of stony desertification areas plays an important role in research on stony desertification [bib_ref] Rocky desertification in Southwest China: Impacts, causes, and restoration, Jiang [/bib_ref]. To better describe the spatial variability, it is of practical significance to accurately visualize the relations between spatial distribution and sampling scale in stony desertification areas. The spatial distribution characteristics of stony desertification in the Houzhai River Basin are closely related to the topography and geomorphology of the watershed, of which there are mainly peaks and low-lying lands in the east, mountains in the north, south and southwest, and mainly plains dotted with a few hills in the middle and west. Corresponding to the topographical and geomorphologic features, stony desertification in this watershed is mainly concentrated in the peaks and low-lying lands in the east, the mountainous areas in the north, south and southwest, as well as the buttes in the middle and west.
## Influencing factors of stony desertification at different sampling scales
The results show that with the increase in the study scale, the spatial correlation of rock exposure changes from moderate to strong; at the same time, in studies at large scales, the correlations between the gradient, elevation and gradient position and the rock exposure are weakened, and rock contents and soil thickness become the key factors influencing stony desertification in studies at large scales. This conclusion has been widely recognized by other experts, and Wang Dian Jie et al. also believe that different topographic factors act on different scales and the impacts of gradient and elevation are mainly manifested at small and medium studies. With the increase in the study scale, the correlation between rock contents and soil properties is more significant. Chen Shengzi et al. believe that the scale variance at large scales increases with fluctuations as the scale increases, and at this moment, stochastic effects have no obvious influences on soil properties.
The gradient and rock exposure of Houzhai River Basin increase with the rise in elevation, while the soil thickness decreases accordingly. It is well known that geographical and climatic conditions at high elevations are poor and not usually suitable for plant growth. However, due to the lack of arable lands, soil at high elevations in the basin is still used for food production, which eventually aggravates the evolution of stony desertification in karst areas. Through a comprehensive analysis, it was found that the gradient, rock exposure, and soil thickness were the main factors that determined the degree of stony desertification [bib_ref] Process of biodiversity research of karst areas in China, Li [/bib_ref]. Stony desertification is a serious problem in the Houzhai River Basin. Gradient and elevation are important factors that lead to soil erosion and stony desertification. The larger the gradient, the more serious the soil erosion caused by overland runoff, thus resulting in stony desertification. As the elevation increases, the environmental conditions become worse, including increases in the gradient and reductions in soil thickness. The vegetation condition becomes worse, and the soil and water conservation capacities at high elevations are relatively weak. In addition, in this study, it is also discovered that the key factors influencing the spatial variability of rock exposure vary with sampling scale. At small scales, the rock exposure is influenced by many factors; at medium scales, it is influenced by gradient, rock contents and rock exposure; and at large scales, the soil thickness and rock exposure are the key factors that influence the spatial variability of rock exposure.
# Conclusions
In this paper, there were some differences in assessment of rock exposure at different scales. The average rock exposure is 15.94% at the scale of 150 m × 150 m, which is the largest, and the average rock exposure is 9.89% at the scale of 900 m × 900 m, which is the smallest. With the increase in sampling scale, the maximum value and the average value of rock exposure decreased gradually while the coefficient of variability increased. At the scale of 150 m × 150 m, the areas of minor stony desertification, medium stony desertification and major stony desertification in the Houzhai River Basin are 7.81 km 2 , 4.50 km 2 , and 1.87 km 2 , respectively. The key factors influencing the spatial variability of rock exposure vary with sampling scale. At small scales (150 m × 150 m, 300 m × 300 m, and 450 m × 450 m), the spatial variability of rock exposure is influenced by gradient, elevation, and soil thickness; at medium scales (600 m × 600 m), it is impacted by gradient and soil gravel content; and at large scales (750 m × 750 m and 900 m × 900 m), soil thickness is the key factor influencing the variability of rock exposure. There is about 22.0 million km 2 of earth surface belonging to the karst landform. This study will present a scientific reference in accurate determination of sampling scale in karst areas.
[fig] Figure 1: The location of Houzhai River basin and the distribution of sample sites. [/fig]
[fig] Figure 2: Plots of soil sample distribution under different sampling scales. [/fig]
[fig] Figure 3: Spatial distribution of rocky desertification under different sampling scales: (a) 150 m × 150 m sampling scale; (b) 300 m × 300 m sampling scale; (c) 450 m × 450 m sampling scale; (d) 600 m × 600 m sampling scale; (e) 750 m × 750 m sampling scale; (f) 900 m × 900 m sampling scale. [/fig]
[table] 105: • 40 43 -105 • 48 2 E, 26 • 12 29 -26 • 17 15 N) is located in Puding County in the central part of Guizhou Province in southwestern China [17], including the three towns of Chengguan (CG), Maguan (MG) and Baiyan (BY), and it covers an area of 75 km 2 . The elevation is between 1223.4 and 1567.4 m.a.s.l., and the air pressure is between 806.1 and 883.8 hpa. There are three major categories of soil: limestone soil, paddy soil and yellow soil. A total of 92.78% of the available flatlands (excluding construction land and watersheds) is used for cropland, and only 3.49% of flatlands are left as various forestland and grassland. The other 3.73% of flatlands consist of uncultivated land. A total of 42.13% of lands on mountains consist of cropland, and 44.95% of lands on mountains are left for various forestland and grassland. A total of 12.95% of lands on mountains consist of uncultivated land due to severe environmental conditions and poor geographical characteristics. The vegetation mainly includes Cupressus funebris Endl., Populus adenopoda Maxim, Toona sinensis (A. Juss.) Roem., Pyrus pyrifolia Burm Nakai., Catalpa bungei C. A. Mey., Pinus massoniana Lamb., and Pyracantha fortuneana (Maxim.) Li. The main crops are Oryzasativa Oryzaglaberrima, Zea mays Linn. Sp., Glycine max (Linn.) Merr, and Helianthus annuus. There are 7 soil types in the study area: Xan Udic Fernalisols, Black Lithomorphic Isohumisols, Cab Udi Orthic Entisols, Cab High-fertility Orthic Anthrosols, Cab Low-fertility Orthic Anthrosols, Cab Medium-fertility Orthic Anthrosols, and Fec Hydragric Anthrosols. [/table]
[table] Table 1: Description and statistics of rock exposures under different sampling scales. [/table]
[table] Table 2: Semi-variance model of rock exposures and its fitting parameters under different sampling scales. [/table]
[table] Table 3: Correlation matrix of rock exposures and its influencing factors under different sampling scales. [/table]
[table] Table 4: Optimal fitted equations of factors influencing rock exposures at different sampling scales. [/table]
|
Plant Variety Protection: Current Practices and Insights
# Introduction
Plant varieties can be improved for numerous agronomic reasons, such as better yields, quality, and resistance to biotic and abiotic stresses. In modern agriculture, crop varieties are derived through plant breeding by mating two or more parental lines that contain desirable characteristics, and the target characteristics are measured over multiple generations under different environmental conditions. Among the progeny, individuals with desirable characteristics are selected, whereas individuals with undesired characteristics are eliminated from the breeding process. This process, repeated over many generations, can create favorable combinations of genetic variations in the next generation, resulting in superior varieties.
The general plant breeding process for both inbreeding and hybrid crops begins with large populations of candidate lines. For example, a commercial corn breeder in the US typically screens approximately 1000 individuals to generate 10-15 individuals in order to introgress the desired characteristic into one competitive genetic background. The 10-15 selected parental plants are cross-pollinated to generate more than 150,000 progeny plants that are evaluated for numerous agronomic characteristics over the course of approximately 6-7 years. More than 99.9% of the progeny plants are eliminated to identify commercially competitive varieties that meet market demand. A breeder usually introgresses several characteristics into multiple backgrounds that have adapted to different environmental conditions. Therefore, this process can result in the screening of hundreds of thousands of individual plants that are grown in hundreds of different environments. Currently, marker-assisted selection can be applied as a two-step process: (1) select progeny carrying the favorable characteristics using markers (trait markers) andscreen and select progeny with those favorable genetic backgrounds using markers (genome-wide markers).
Breeding new varieties is a resource-intensive activity in terms of costs, infrastructure, genetic resources, and the breeders' knowledge and experience. It can also be timeconsuming: it typically takes around six to seven years to release a new variety but can take up to ten years. A video recording on the costing of breeding programs in the public and private sectors can be viewed at https://www.youtube.com/watch?v=x8QXauBzhYo, accessed on 21 July 2021. However, plant varieties can be reproduced easily and quickly, and breeders need to secure their investment return. Plant breeders have various options to protect their ownership of new varieties, prevent outlawed practices and recover breeding costs from royalties. Such options motivate breeders and support further breeding activities that constantly provide farmers with the best varieties that satisfy consumer demand.
## Protection of the breeder's right
Plant variety protection (PVP) via the International Union for the Protection of New Varieties of Plants (UPOV) is a harmonized system that awards intellectual property rights (IPR) to organizations in its 75 member states (as of February 2019; https://www.upov. int/portal/index.html.en, accessed on 21 July 2021). It recognizes both the plant breeder's right (PBR) and the plant variety right (PVR). Under the 1991 Act of the UPOV Convention, the PBR is granted for a period of no less than 20 years. Under the UPOV system, anyone can be a breeder and can apply for PVP. However, a new plant variety can only be protected if it meets four criteria established by the UPOV: (1) it must be a novel variety, (2) it must be distinct from other varieties, (3) it must display homogeneity, and (4) it must be stable, remaining unchanged from one generation to the next. Only the breeder can obtain protection for a new variety. A few examples of activities that can be considered infringements if they are carried out without the certified owner's permission are (1) the sale and marketing of a protected variety, (2) sexual multiplication or unauthorized propagation, (3) use of parental lines for hybrid variety production, and (4) distribution of the variety without the permission of the certified owner. Under the 1991 Act of the UPOV Convention, there are a few exceptions to the breeder's right, also known as breeder's exemption: the right of all to use protected varieties as sources of initial variation, the right of all to use protected varieties for research or experimental purposes, and the right of all to use protected varieties for private, non-commercial purposes (e.g., amateur gardeners).
In some countries, such as the USA, Japan, and Australia, breeders can also apply for plant or utility patent rights if the new varieties satisfy the required criteria. The Plant Variety Protection Office (PVPO) in the US provides intellectual property protection for breeders of new varieties of seeds, tubers, and asexually propagated plants (https: //www.ams.usda.gov/services/plant-variety-protection, accessed on 21 July 2021). There are three types of intellectual property protection: (1) PVP issued by the PVPO, (2) plant patents issued by the Patent and Trademark Office (PTO) for asexually propagated plants (except edible tubers), and (3) utility patents issued by the PTO for genes, traits, methods, plant parts, or varieties. This article will focus on PVP under the UPOV system. The authors will seek another opportunity to address different types of PVP, such as variety patents, utility patents, trade secrets, and more.
These protection systems aim to give the owners of plant varieties exclusive rights to control production, distribution, and marketing and assure that owners are compensated for the costs of developing new varieties.
## The distinctness, uniformity, and stability (dus) test
Within the UPOV's PVP framework, all members acknowledge breeders of new varieties of plants by granting them intellectual property rights based on the following conditions: distinctness (D) from existing varieties, uniformity (U) of relevant characteris-tics that depend on the reproduction system of the species, and stability (S) in expression of traits after two independent growing cycles. Details of these conditions can be accessed from https://www.upov.int/overview/en/conditions.html, accessed on 21 July 2021. The system for establishing distinctness, uniformity, and stability is known as the DUS test (UPOV 1991). Therefore, before applying for PVP, applicants should have bred a new variety, completed the DUS test, and chosen a unique name for it.
The following sections discuss methods of proving DUS-phenotypes-based, molecular markers-based, and sequencing-based tests-and their limitations, practicable solutions, and potentialities.
Genes 2021, 12, x FOR PEER REVIEW 3 of 12
## The distinctness, uniformity, and stability (dus) test
Within the UPOV's PVP framework, all members acknowledge breeders of new varieties of plants by granting them intellectual property rights based on the following conditions: distinctness (D) from existing varieties, uniformity (U) of relevant characteristics that depend on the reproduction system of the species, and stability (S) in expression of traits after two independent growing cycles. Details of these conditions can be accessed from https://www.upov.int/overview/en/conditions.html, accessed on 21 July 2021. The system for establishing distinctness, uniformity, and stability is known as the DUS test (UPOV 1991). Therefore, before applying for PVP, applicants should have bred a new variety, completed the DUS test, and chosen a unique name for it. The following sections discuss methods of proving DUS-phenotypes-based, molecular markers-based, and sequencing-based tests-and their limitations, practicable solutions, and potentialities.
[formula] (a) (b) (c) [/formula]
## Phenotypes-based dus test
The current PVP system relies on the morphological description of plant varieties. The DUS test determines whether a new variety is distinguishable from commonly known varieties and exhibits sufficient phenotypic uniformity and stability. A DUS test is normally conducted in a field or greenhouse over two successive growing seasons at two test locations. During this period a number of morphological characteristics are recorded for both the new (or candidate) variety and similar varieties, in what is known as common knowledge. The DUS test, whether it is carried out in a trial field or greenhouse, must follow internationally agreed protocols and UPOV guidelines. Over 300 test guidelines are currently available for different plant species; these guidelines can be accessed from the UPOV website at https://www.upov.int/resource/en/dus_guidance.html, accessed on 21 July 2021.shows the list of characteristics used for testing corn, wheat, rice, barley, and soybean among others.
## Phenotypes-based dus test
The current PVP system relies on the morphological description of plant varieties. The DUS test determines whether a new variety is distinguishable from commonly known varieties and exhibits sufficient phenotypic uniformity and stability. A DUS test is normally conducted in a field or greenhouse over two successive growing seasons at two test locations. During this period a number of morphological characteristics are recorded for both the new (or candidate) variety and similar varieties, in what is known as common knowledge. The DUS test, whether it is carried out in a trial field or greenhouse, must follow internationally agreed protocols and UPOV guidelines. Over 300 test guidelines are currently available for different plant species; these guidelines can be accessed from the UPOV website at https://www.upov.int/resource/en/dus_guidance.html, accessed on 21 July 2021.shows the list of characteristics used for testing corn, wheat, rice, barley, and soybean among others.
The phenotype-based DUS test is a time-consuming, laborious (frequently requires skill and adequate experience), and costly process of field and greenhouse assessment. In some cases, special conditions or trial designs are required to conduct the tests, for example, vernalization in barley and wheat. In addition, as the numbers of candidates and publicly known cultivars increase, the ability to distinguish them on the basis of morphological traits alone becomes more difficult, even though differences in agronomic performance may exist.
## Limitations of the conventional phenotypes-based dus test results
In the current system, many factors limit the use of morphological traits to conduct a DUS test. The measurement of morphological traits may be influenced by environmental factors, including locations and time spans, which may complicate the assessment. These factors can affect the expression of morphological traits, resulting in reduced precision and discriminatory power. This is especially disadvantageous for the evaluation of disease-resistant traits, which are mandatory for the majority of crops, or complex traits (known as quantitative traits), such as drought resistance and flowering time.
In a backcross breeding program, the top-performing variety (existing protected variety) is often repeatedly crossed with other breeding lines. This requires ongoing evaluation and selection of progeny with the most desired agronomic characteristics and removal of any progeny with undesired characteristics. By the sixth backcross, the selected progeny will theoretically contain more than 99% of the genetic background of the topperforming varieties, but less than 1% of the other breeding lines. There are other breeding techniques, such as mutation breeding and genetic engineering, that can also create minor genetic modifications. In these cases, a new variety would be considered to be an essentially derived variety (EDV) from the initial variety (IV). The essential derivation and dependency concepts have also been defined by the 1991 Act of the UPOV Convention; with this process, most of the IV's essential characteristics remain intact in the newly bred variety. For example, if a new variety meets the criteria of practically containing the entire genotype of a variety from which it was derived, and therefore, genetically maintains the expression of the IV's essential traits, the newly bred variety is considered to be an EDV from the IV. Therefore, EDVs share their essential traits with the IV, except their distinctive traits. To determine EDV, or distinguish between EDV and fraud, the criteria and procedures used for phenotype-based DUS tests are often insufficient or not applicable because there will always be important morphological traits that differentiate an EDV from the IV.
The increased availability and cost-efficiency of molecular markers make them appealing options that supplement existing phenotype-based DUS tests. Molecular markers-based DUS tests have several notable advantages over phenotype-based DUS tests, such as high discriminatory power, high repeatability, independence from environmental effects, applicability to seed or early growth stages of plants, rapid data production and analysis, and amenability to readily searchable databases containing records of thousands of varieties, which can also facilitate global harmonization. In addition, to characterize EDVs, molecular markers can be used to conduct tests based on the genetic relatedness of IVs and EDVs.
## Molecular markers-based dus test
The UPOV's Working Group on Biochemical and Molecular Techniques and DNA-Profiling in Particular (BMT) consists of biochemical and molecular DUS testing experts and plant breeders (UPOV 2013; https://www.upov.int/edocs/tgpdocs/en/tgp_15.pdf, accessed on 21 July 2021). The purpose of the UPOV-BMT is to explore practical aspects, such as the application of molecular markers for plant-variety registration, and to strengthen the protection afforded by the PBR. So far, the BMT has proposed two models. Model 1 is the use of molecular markers that are directly linked to traditional and molecular characteristics as predictors of traditional characteristics. It requires prerequisite research to identify marker-trait associations that maintain their robustness across diverse germplasm. Model 2 is the calibration of threshold levels for molecular markers against the minimum distance in phenotype traits. This application potentially provides the basis for the introduction of a system for combining phenotypic and molecular distances in the management of variety collections as a tool to improve the efficiency of distinctness evaluation.
Due to the abundance of reference genomes and advances in molecular technologies, such as gene cloning, markers that target the specific alleles responsible for phenotype variation, the so-called functional markers (FM), can now be developed. FMs could be highly predictive of trait expression and overcome the problem of using flanking markers to the traits that are prone to recombination between the marker and the actual target alleles.
Various studies have demonstrated the use of markers for the assessment of distinctness. Cockram et al.tested 25 single nucleotide polymorphism (SNP) markers for 14 DUS traits on a panel of 169 barley varieties. Three DUS traits (ear (number of rows), grain (disposition of lodicules), and seasonal growth habit) were perfectly predicted by the genotype data. The remaining traits, ranging from 81% to 91%, were also predicted by the genotype data. With 41 corn inbred lines, simple sequence repeat (SSR) markers showed better performance than DUS traits in distinguishing and fitting their pedigree information. Sarao et al.demonstrated that molecular-marker data differentiated japonica and indica rice subspecies. SSR markers, using cluster and principal component analysis (PCA), distinguished basmati from non-basmati groups and showed congruency with the pedigree and breeding history of 14 rice varieties. These results suggest that the prediction value and genetic distance calculated on the markers revealed better consistency and correlated with the pedigree of varieties.
Wang et al.established a method of applying molecular markers for the assessment of stability. A set of 347 SSR markers was genotyped using 20 wheat breeding lines and the homozygous SSR loci ratio (SSR-HLR) was calculated to identify non-homozygous SSR loci. By comparing the morphological observation with the SSR-HLR, more than 95% of varieties were considered stable, and less than 91% were considered unstable. The varieties with SSR-HLR ranging from 91% to 95% were suggested for morphological observations as the field condition for stability assessment. This approach has several advantages: better selection of relevant references in field trials, exclusion of non-relevant reference varieties from field trials, reduction in the number of reference varieties in field trials, and reduction in the duration of field trials, which leads to a reduction in overall costs and resources.
In cases where molecular data provide greater discrimination than phenotypic comparisons, an important question needs to be asked when using molecular markers in DUS tests: how different is different? Breeders have mentioned that the use of marker data in DUS evaluation might lead to the introduction of unrealistically and unnecessarily high levels of uniformity being required at the DNA sequence level, which may lead to higher resource demands during breeding and seed multiplication. Regardless of data sources, whether morphological, physiological, or molecular markers, determining a distinctness threshold may lead to the question of how to define minimum distance in order to maintain the current level of intellectual property protection.
Archard et al.compared the SNP-based similarities and pedigree-based kinships of 322 closely related PVP-certified soybean varieties. The initial analyses suggest there is evidence of distinctness where SNP similarity is in the range of 93-97%. A subsequent analysis suggests that 96% SNP similarity could provide an acceptable threshold for the assessment of distinctness. Consequently, varieties that are more than 96% similar according to SNP marker data, but also differ in their morphological or physiological traits, would continue to be classified as distinct for as long as these traits are the ultimate test of distinctness. Archard et al.measured the intracultivar heterogeneity of SNP loci of 35 commercially developed soybean varieties. The levels of intracultivar SNP heterogeneity were low (range 0-5%, mean 1.8%, and standard deviation 1.3%) for all 35 varieties. These levels of SNP heterogeneity are consistent with the commonly used breeding strategy of bulking individual plants at the F4 stage of inbreeding.
Van Eeuwijk and Lawdiscussed the criteria to select markers when developing an EDV protocol, including marker unbiasedness, precision, and genome coverage. The distribution of genetic similarities between corn parental inbred lines and their progeny derived from F2 and different backcross populations was examined to determine whether markers should be evenly distributed across the entire genome or be confined to specific regions that control differences in DUS traits. The results show that increasing the number and length of chromosomes when using abundant and evenly distributed markers leads to a decrease in standard deviations, which in turn increases prediction accuracy.
## Sequencing-based dus test
There are technical challenges for molecular markers-based assessment. Even though functional markers (FM) are becoming more abundant, the majority of molecular markers applied to breeding programs are not directly linked to traits. Certain genomic areas of trait that are of interest are located in highly repetitive genome areas, which makes the design of markers difficult. In addition, there are other types of genomic variants, such as structural variants (SVs) and copy number variations (CNV) that are also directly responsible for traits. Identification of such causative variants has been inhibited due to the limitations of common marker technologies, such as SNP and SSR. Due to the rapid cost reductions of next-generation sequencing (NGS) and genotyping-by-sequencing (GBS) platforms, plant breeders are switching from conventional marker technology to sequencing technology. Various NGS technologies are being applied to breeding programs, for example, short-read whole-genome sequencing (WGS), and targeted NGS or targeted GBS.
Targeted NGS allows the sequencing of a specific genomic region in order to detect causative variants more rapidly and cost-effectively than WGS. Targeted NGS may become an attractive replacement for the conventional marker technology that is used for markerbased DUS testing. CRISPR-Cas9 -enriched long-read sequencing (LRS) is a good example of targeted NGS. By directing CRISPR-Cas9 for targeted cleavage in order to isolate a region of interest, followed by enrichment and LRS, it is possible to excise long, intact DNA molecules from the trait of interest. This allows the target region to be isolated without amplification and subsequently investigated using LRS technologies. The resulting long-read data not only give a comprehensive view of the region of interest and make it possible to identify complex structural variation but also repeat elements that are difficult to find with other DNA-sequencing technologies.
## Noteworthy subjects in addition to the current upov and dus tests
By definition of the UPOV convention, conventional breeding generally relies on genetic recombination. The process enables the selection of superior varieties by creating new allelic and phenotypic combinations. With advances in new breeding technologies (NBTs), plant breeders could manipulate gene sequences precisely in order to create new favorable alleles or remove unfavorable ones. This technology, known as genome editing, may revolutionize plant breeding, leading to the development of new varieties. Among the various genome editing tools, CRISPR is becoming popular because of its high efficiency and simplicity. It modifies genotypic distances between varieties through genome editing, and the modification can occur at the level of a few base-pair nucleotides. However, this leads to some controversy. If the IV is the variety protected by the PBR, the new genome-edited variety would be subject to an EDV claim since it wholly conforms and retains the essential characteristics of the IV except for a few base-pair nucleotides. This minor modification adds significant agronomic value since herbicide resistance by the IV could also be considered a distinctive trait in DUS testing. Legal clarification on the definition of EDV is of fundamental importance in order to avoid or discourage active adaption of NBT in breeding programs. Therefore, redefinition of the EDV concept is inevitable, and harmonized guidelines on what is publicly and internationally accepted are required by next-generation breeders.
In the EU, a mandatory variety-testing system is used in addition to DUS testing before new varieties are released (value for cultivation and use (VCU) testing). Where DUS traits focus on morphological characteristics, VCU focuses on more agronomic characters. There are four categories for VCU testing: (1) yield; (2) resistance to biotic and abiotic stresses; (3) end-use quality, such as malting quality in barley, protein content in wheat, and oil quality/quantity in rapeseeds; and (4) factors in the physical condition, such as susceptibility to damage. Some candidate varieties with proven superior VCU fail DUS even though the non-distinct comparison is made with a significantly lower-performing registered cultivar. To resolve this case, an additional value molecular-linked DUS (vmDUS) distinctness tool that uses molecular markers, but conforms to UPOV-declared principles, has been proposed.
The open source seed initiative (OSSI) was initiated in 2012, inspired by the open source software movement. The OSSI emerged in the United States, where patenting of plant varieties is common. OSSI wants to create a system that can go viral: IVs would be freely available to breeders under the condition that any genetic resources (varieties) derived from them would be made available under the same open source conditions. This would be achieved through a pledge (in the USA). The F1 hybrid can be OSSI-pledged with the information from parental lines involved in the cross to produce the hybrid, but the parental lines will not be required to be OSSI-pledged or available (https://osseeds.org/faqs/, accessed on 21 July 2021). In 2014, 37 varieties of 14 species were released under the OSSI-Pledge by various breeders in the public and private sectors. PVP has been developed in order to stimulate invention and is intended to support the use of inventions in practical innovations that positively impact society. There is some concern that the OSSI movement may discourage innovation and that it may eventually negatively impact societal benefits. Finding a balance between breeders' rights and obligations is complex in a society where rapidly advancing technology increasingly alienates the inventor from society at large. OSSI should consider a way of providing sufficient innovation that requires significant investments in plant breeding that contribute to societal goals, including the provision of more robust plant varieties that meet farmers' and end-user customers' demands.
## Closing remark
Advances in genotyping and sequencing platforms, and reductions in their usage costs, provide opportunities for their potential application in variety registration. Chip and array technologies, consisting of hundreds of thousands of SNP markers, are being actively applied to breeding programs. In addition, advances in statistical methodologies and increased computing power have led to genome-wide, low-passing sequencing technology being rapidly adopted by breeding programs. However, none of these have been officially adopted by the UPOV. The UPOV currently requires the use of molecular markers only when they perfectly correlate with DUS traits, which does not reflect the advances in genotyping and sequencing technologies or an understanding of DUS trait genetics. Authors should urge the BMT working group to kick off another round of technical discussions related to the recognition of the potential of these technologies, the exploration of new opportunities, and the prompt adoption of these technologies for next-generation DUS testing.
Herein, the overall cost estimation of PVP, including DUS testing, is not addressed. One of the main reasons is that the current breeding operation adoption level and molecular technologies vary by public vs. industry, well-funded institutes vs. poorly funded, and major grain crops vs. vegetables. For example, if a breeder works in a well-funded institute or industry, all DUS characteristics and molecular marker information can be gathered during the line development stage. Then, this breeder (or institute) may only need resources to cover the cost of registration and maintenance of the PVP. However, if a breeder works in an older or poorly funded breeding environment, the cost of gathering DUS characteristics is incredibly challenging. Therefore, the authors consider that cost estimation of the PVP process may not be relevant and desire to avoid any possibility of misinterpretation of the information by readers.
As discussed, new breeding technologies, such as genome editing, are common in both the public and private sectors. Consideration of a new definition of plant variety is a priority for those involved in the development, evaluation, and release of new crop varieties. Plant variety patenting is common practice in the US, for example. VCU testing is now a mandatory step, in addition to DUS testing, before variety registration in the EU. Procedures and technical methods for plant variety testing require significant, harmonized international standards that create the basis for strengthening the supply of improved varieties and the PVP system. Therefore, as an intergovernmental organization, the UPOV's direct involvement and significant contribution to these areas are urged.
When a PVP certificate officially expires (exPVP), and the active variety patent is no longer valid, the original variety enters the public domain. With advanced high-throughput, high-density genotyping and sequencing technologies combined with advanced statistical mythologies and high computing power, exPVP lines potentially represent a new germplasm source for any breeding program. It has also opened new opportunities to discover and exploit the genes controlling agronomic performance in conventional breeding programs and genome editing programs. As mentioned earlier, the authors plan for another manuscript that will focus on variety protection beyond PVP under the UPOV system, like variety patents, utility patents, trade secrets, and how public and private sectors utilize exPVP lines for their breeding and research programs. |
Organization of the BcgI restriction-modification protein for the cleavage of eight phosphodiester bonds in DNA
Type IIB restriction-modification systems, such as BcgI, feature a single protein with both endonuclease and methyltransferase activities. Type IIB nucleases require two recognition sites and cut both strands on both sides of their unmodified sites. BcgI cuts all eight target phosphodiester bonds before dissociation. The BcgI protein contains A and B polypeptides in a 2:1 ratio: A has one catalytic centre for each activity; B recognizes the DNA. We show here that BcgI is organized as A 2 B protomers, with B at its centre, but that these protomers self-associate to assemblies containing several A 2 B units. Moreover, like the well known FokI nuclease, BcgI bound to its site has to recruit additional protomers before it can cut DNA. DNA-bound BcgI can alternatively be activated by excess A subunits, much like the activation of FokI by its catalytic domain. Eight A subunits, each with one centre for nuclease activity, are presumably needed to cut the eight bonds cleaved by BcgI. Its nuclease reaction may thus involve two A 2 B units, each bound to a recognition site, with two more A 2 B units bridging the complexes by protein-protein interactions between the nuclease domains.
# Introduction
Restriction-Modification (RM) systems use two enzyme activities to defend bacteria against foreign DNA, a restriction endonuclease (REase) and a modification methyltransferase (MTase) [bib_ref] Immigration control of DNA in bacteria: self versus non-self, Murray [/bib_ref]. The MTase transfers a methyl group from S-adenosylmethionine (SAM) to a cytosine or an adenine within a particular DNA sequence, the recognition site for that system, while the REase cleaves DNA with unmethylated sites [bib_ref] REBASE-a database for DNA restriction and modification: enzymes, genes and genomes, Roberts [/bib_ref]. The majority of RM systems fall into either the Type I or Type II categories [bib_ref] A nomenclature for restriction enzymes, DNA methyltransferases, homing endonucleases and their genes, Roberts [/bib_ref]. Most Type I systems are oligomeric (R 2 M 2 S) proteins containing subunits for DNA cleavage (R), methylation (M) and sequence specificity (S) [bib_ref] Nucleoside triphosphate-dependent restriction enzymes, Dryden [/bib_ref] [bib_ref] Structure and operation of the DNA-translocating Type I DNA restriction enzymes, Kennaway [/bib_ref] , though in some cases the R, M and S functions are all carried as domains within a single polypeptide [bib_ref] DNA cleavage and methylation specificity of the single polypeptide restriction-modification enzyme LlaGI, Smith [/bib_ref]. The cleavage activity is triggered by unmodified sites, though it often occurs distant from the site, due to the R subunits possessing an ATP-dependent DNA translocase. The main reaction of the MTase is to convert hemi-methylated sites, methylated in one strand only, to fully modified products methylated in both strands. Neither the fully nor the hemi-methylated DNA is cut by the REase. Consequently, DNA fully modified once by the MTase is never cleaved by the REase even after its semi-conservative replication.
Most Type II RM systems use separate proteins for restriction and modification that both bind to the same DNA sequence [bib_ref] AdoMet-dependent methylation, DNA methyltransferases and base flipping, Cheng [/bib_ref] [bib_ref] Type II restriction endonucleases: structure and mechanism, Pingoud [/bib_ref]. The REase is commonly a homodimer that recognizes a palindromic sequence and cuts both strands at specified loci within the site: one subunit attacks the target phosphodiester bond in one strand, while the other cuts the scissile bond in the opposite strand [bib_ref] Type II restriction endonucleases, Perona [/bib_ref]. The MTase is usually a monomer whose main role is to transfer one methyl group onto DNA already methylated in one strand [bib_ref] AdoMet-dependent methylation, DNA methyltransferases and base flipping, Cheng [/bib_ref]. But many Type II systems deviate considerably from these classical systems. They have been categorized into numerous subtypes-IIG, IIE, IIF, IIS and IIB, among others-on the basis of their genetic organization, their mode of action and their recognition sites and cleavage loci [bib_ref] A nomenclature for restriction enzymes, DNA methyltransferases, homing endonucleases and their genes, Roberts [/bib_ref].
Perhaps the only aspect common to all Type II REases, and the one that differentiates them from Type I systems, is that they cleave DNA at fixed positions relative to their recognition sites.
Variations in genetic organization include the Type IIG systems that, instead of separate REase and MTase proteins, possess a single polypeptide with both activities [bib_ref] Purification and properties of the Eco57I restriction endonuclease and methylase -prototypes of..., Janulaitis [/bib_ref] [bib_ref] Characterization of BcgI, a new kind of restriction-modification system, Kong [/bib_ref] [bib_ref] The HaeIV restriction modification system of Haemophilus aegyptius is encoded by a..., Piekarowicz [/bib_ref] [bib_ref] MmeI: a minimal Type II restriction-modification system that only modifies one DNA..., Morgan [/bib_ref]. The IIG systems require SAM not only as a co-factor for their methylation reactions but also to activate the nuclease.
Variations in mode of action are illustrated by the Type IIE and IIF systems [bib_ref] Diversity of Type II restriction endonucleases that require two DNA recognition sites, Mucke [/bib_ref] [bib_ref] Enzyme-mediated DNA-looping, Halford [/bib_ref]. While the orthodox enzymes act at individual sites, the IIE and IIF REases need two copies of their target sequence for full activity. The IIE enzymes, often dimers, proceed to cut both DNA strands at one of the two sites; the other site acts as an allosteric activator [bib_ref] Nonidentical DNA-binding sites of endonuclease NaeI recognize different families of sequences flanking..., Yang [/bib_ref] [bib_ref] DNA cleavage reactions by type II restriction enzymes that require two copies..., Embleton [/bib_ref]. In contrast, the Type IIF enzymes tend to cut both strands at both sites before leaving the DNA [bib_ref] DNA cleavage reactions by type II restriction enzymes that require two copies..., Embleton [/bib_ref] [bib_ref] The SfiI restriction endonuclease makes a four-strand DNA break at two copies..., Wentzell [/bib_ref] : most operate as tetramers of identical subunits, with the four active sites, one in each subunit, each positioned to cleave one of the four target bonds [bib_ref] Structure of the tetrameric restriction endonuclease NgoMIV in complex with cleaved DNA, Deibert [/bib_ref] [bib_ref] A view of consecutive binding events from structures of tetrameric endonuclease SfiI..., Vanamee [/bib_ref] [bib_ref] DNA synapsis through transient tetramerization triggers cleavage by Ecl18kI restriction enzyme, Zaremba [/bib_ref]. Enzymes that require two DNA sites can be identified by comparing their activities on DNA substrates with one or two cognate sites [bib_ref] The SfiI restriction endonuclease makes a four-strand DNA break at two copies..., Wentzell [/bib_ref] [bib_ref] DNA synapsis through transient tetramerization triggers cleavage by Ecl18kI restriction enzyme, Zaremba [/bib_ref] [bib_ref] Many type IIs restriction endonucleases interact with two recognition sites before cleaving..., Bath [/bib_ref] [bib_ref] Restriction endonucleases that bridge and excise two recognition sites from DNA, Marshall [/bib_ref]. Proteins that interact with two sites prefer sites in cis, where they loop out the intervening DNA, over sites in trans, where they must hold together two separate DNA molecules [bib_ref] Enzyme-mediated DNA-looping, Halford [/bib_ref]. Consequently, both Type IIE and IIF REases can cleave two-site substrates more rapidly than a one-site DNA. However, IIE enzymes cut two-site substrates one site at a time, whereas IIF enzymes cut both sites together [bib_ref] DNA cleavage reactions by type II restriction enzymes that require two copies..., Embleton [/bib_ref].
Variations in recognition and cleavage loci are shown by the Type IIS REases, which bind asymmetric rather than palindromic sites and which cut the DNA at fixed loci away from the sequences, on one side of it [bib_ref] Class-IIS restriction enzymes -a review, Szybalski [/bib_ref] [bib_ref] Natural and engineered nicking endonucleases -from cleavage mechanism to engineering of strand-specificity, Chan [/bib_ref]. For example, FokI recognizes 5 0 À GGATG À N ð Þ 9 # À3 0 3 0 À CCTAC À N ð Þ 13 " À5 0 and cuts both strands at the positions indicated (#,"), 9 and 13 nt away [bib_ref] REBASE-a database for DNA restriction and modification: enzymes, genes and genomes, Roberts [/bib_ref]. An asymmetric sequence cannot be recognized in the same way as a dimeric enzyme recognizing a palindromic site, where each subunit contacts one half of the site [bib_ref] Type II restriction endonucleases, Perona [/bib_ref]. Instead, either a monomeric protein contacts the entire site, as is the case with FokI [bib_ref] Structure of the multimodular endonuclease FokI bound to DNA, Wah [/bib_ref] , or different subunits in an oligomeric protein each contact discrete parts of the sequence, as with BbvCI [bib_ref] Site-specific DNA-nicking mutants of the heterodimeric restriction endonuclease R, Heiter [/bib_ref]. If the nuclease is a monomer, it may have only one active site [bib_ref] Natural and engineered nicking endonucleases -from cleavage mechanism to engineering of strand-specificity, Chan [/bib_ref] , yet it needs to cut both 5 0 -3 0 and 3 0 -5 0 strands. The use of the same active site to cut both strands is often-but not always-prohibited, as it requires the site to rotate not only around the DNA, from one strand to the other, but also perpendicular to the DNA, to match their antiparallel nature [bib_ref] A novel mechanism for the scission of double-stranded DNA: BfiI cuts both..., Sasnauskas [/bib_ref]. To cut both strands, the monomer of FokI bound to the DNA associates with a second monomer [bib_ref] FokI dimerization is required for DNA cleavage, Bitinaite [/bib_ref] [bib_ref] Protein assembly and DNA looping by the FokI restriction endonuclease, Catto [/bib_ref] [bib_ref] Illuminating the reaction pathway of the FokI restriction endonuclease by fluorescence resonance..., Pernstich [/bib_ref] to give a dimeric unit with two active sites [bib_ref] Structure of FokI has implications for DNA cleavage, Wah [/bib_ref]. The second active site can alternatively come from the isolated catalytic domain of the FokI protein, without its DNA recognition domain [bib_ref] FokI dimerization is required for DNA cleavage, Bitinaite [/bib_ref]. The two catalytic centres each cut one specified strand: the DNA-bound monomer, the bottom strand 13 nt away and the recruited unit, the top strand 9 nt distant [bib_ref] Targeting individual subunits of the FokI restriction endonuclease to specific DNA strands, Sanders [/bib_ref]. However, the enzyme at the site associates more readily with another monomer bound elsewhere in the same DNA than with one in free solution, as proteins in cis cannot escape from each other [bib_ref] Protein assembly and DNA looping by the FokI restriction endonuclease, Catto [/bib_ref] [bib_ref] DNA looping by FokI: the impact of synapse geometry on loop topology..., Rusling [/bib_ref]. FokI thus attacks two-site substrates more rapidly than one-site DNA, although the two-site substrate is initially cut at just one site: the residual site is then cleaved at the same slow rate as the one-site DNA [bib_ref] Many type IIs restriction endonucleases interact with two recognition sites before cleaving..., Bath [/bib_ref] [bib_ref] Protein assembly and DNA looping by the FokI restriction endonuclease, Catto [/bib_ref]. The need for two recognition sites is common among the Type IIS REases: some, like FokI, act as IIE enzymes; others, like the tetramer BspMI, as IIF enzymes [bib_ref] The type IIs restriction endonuclease BspMI is a tetramer that acts concertedly..., Gormley [/bib_ref]. Another group that cleave outside their recognition sites are the Type IIB systems [bib_ref] The Type IIB restriction endonucleases, Marshall [/bib_ref]. Unlike the IIS enzymes that cut the DNA on one side of the site, the nucleases from the Type IIB systems make two double-strand breaks (DSBs) at each site, one either side, to excise a short fragment that carries the recognition sequence. Moreover, their recognition sequences usually consist of two specified but divergent blocks of sequence separated by a non-specific segment of fixed length, like many Type I sites [bib_ref] Nucleoside triphosphate-dependent restriction enzymes, Dryden [/bib_ref]. For example, BcgI, an archetype of the IIB enzymes [bib_ref] Characterization of BcgI, a new kind of restriction-modification system, Kong [/bib_ref] [bib_ref] A unique restriction endonuclease, BcgI, from Bacillus coagulans, Kong [/bib_ref] [bib_ref] Analyzing the functional organization of a novel restriction modification system, the BcgI..., Kong [/bib_ref] [bib_ref] Does BcgI, a unique restriction endonuclease, require two recognition sites for cleavage?, Kong [/bib_ref] , recognizes the sequence, 5 0 À # 10 ðNÞ À CGA À ðNÞ 6 À TGC À ðNÞ 12 # À3 0 3 0 À " 12 ðNÞ À GCT À ðNÞ 6 À ACG À ðNÞ 10 " À5 0 Its REase cuts both strands on both sides of this sequence, 10 and 12 nt away, to leave the site on a fragment 34 nt long in both strands (noted below as the 34-mer): its MTase targets the adenines marked above in italics. However, the BcgI nuclease has no activity when bound to a solitary site [bib_ref] Concerted action at eight phosphodiester bonds by the BcgI restriction endonuclease, Marshall [/bib_ref]. Instead, like most Type IIB REases [bib_ref] Restriction endonucleases that bridge and excise two recognition sites from DNA, Marshall [/bib_ref] , it needs two sites [bib_ref] Does BcgI, a unique restriction endonuclease, require two recognition sites for cleavage?, Kong [/bib_ref] , ideally in cis but alternatively in trans. After bridging the sites, it then proceeds to make two DSBs at both sites before releasing the final products, thus cutting a total of eight phosphodiester bonds within a single complex: hardly any of the partially cleaved intermediates accumulate during the course of the reaction [bib_ref] Concerted action at eight phosphodiester bonds by the BcgI restriction endonuclease, Marshall [/bib_ref].
BcgI, like many but not all Type IIB systems [bib_ref] The Type IIB restriction endonucleases, Marshall [/bib_ref] , is composed of two subunits: an A subunit of 71.6 kDa that carries both MTase and REase catalytic functions; a B subunit of 39.2 kDa that recognizes the target sequence [bib_ref] Characterization of BcgI, a new kind of restriction-modification system, Kong [/bib_ref] [bib_ref] Analyzing the functional organization of a novel restriction modification system, the BcgI..., Kong [/bib_ref]. Like the Type IIG systems that have both activities in the same subunit, BcgI requires SAM not only for its MTase but also for its REase function: the nuclease also needs Mg 2+ [bib_ref] A unique restriction endonuclease, BcgI, from Bacillus coagulans, Kong [/bib_ref]. Densitometry of coomassie-stained SDS gels yielded a 2:1 ratio of A:B subunits [bib_ref] Characterization of BcgI, a new kind of restriction-modification system, Kong [/bib_ref] , albeit with the assumption-not always valid (42)-that the two polypeptides stain to the same extent. However, size exclusion chromatography of native BcgI gave apparent MW values of 250-300 kDa [bib_ref] Analyzing the functional organization of a novel restriction modification system, the BcgI..., Kong [/bib_ref] , larger than the 182 kDa expected for a protein with one B and two A subunits. Nevertheless, the size exclusion MW app can be related to an A 2 B protomer if that unit has a large frictional ratio or if it self-associates.
The A subunit of BcgI resembles a fusion of R and M subunits from a Type I system without the ATP motor functions of R, while B is strikingly similar to an S subunit: it likewise possesses separate domains for recognizing each section of its bipartite target site [bib_ref] Analyzing the functional organization of a novel restriction modification system, the BcgI..., Kong [/bib_ref]. An A 2 B assembly for BcgI is thus a direct analogue of the R 2 M 2 S organization of Type I systems. Other Type IIB systems contain a single subunit with all three functions [bib_ref] The HaeIV restriction modification system of Haemophilus aegyptius is encoded by a..., Piekarowicz [/bib_ref] [bib_ref] Characterization of AloI, a restriction-modification system of a new type, Cesnaviciene [/bib_ref] , akin to the single-polypeptide Type I enzymes [bib_ref] DNA cleavage and methylation specificity of the single polypeptide restriction-modification enzyme LlaGI, Smith [/bib_ref]. But while Type I REases generally make one DSB, usually about halfway between two recognition sites, BcgI makes four DSBs per reaction [bib_ref] Concerted action at eight phosphodiester bonds by the BcgI restriction endonuclease, Marshall [/bib_ref]. How the BcgI RM protein is organized to hydrolyse eight phosphodiester bonds in a complex with two recognition sites is addressed here. In the following article [bib_ref] Organisation of the BcgI restriction-modification protein for the transfer of one methyl..., Smith [/bib_ref] , we show that the BcgI MTase transfers to an individual site in the DNA just one methyl group at a time. Hence, this protein may be organized differently for its REase and MTase activities.
# Materials and methods
## Dna
The plasmid pARG3-BcgI (a gift from New England Biolabs) over-expresses the native forms of bcgIA and bcgIB to yield the wild-type (WT) BcgI protein. The plasmid was subjected to site-directed mutagenesis (QuikChange II: Agilent) to replace Glu53 in the A subunit with Ala, to give pARG3-BcgIE53A [the E53A mutant has no nuclease activity [bib_ref] Analyzing the functional organization of a novel restriction modification system, the BcgI..., Kong [/bib_ref] ]. To construct the plasmid pAJJ1, the section of pARG3-BcgI encompassing bcgIA was amplified by PCR and the resultant fragment cloned between the NcoI and XhoI sites of pET28a (Merck).
The supercoiled (SC) forms of the plasmids pUC19 and pDG5 were 3 H-labelled and purified as described previously [bib_ref] The SfiI restriction endonuclease makes a four-strand DNA break at two copies..., Wentzell [/bib_ref] [bib_ref] Restriction endonucleases that bridge and excise two recognition sites from DNA, Marshall [/bib_ref]. The following duplexes, all with HEX (hexachlorofluorescein) at the 5 0 -end of the top strand, were as before (41): HEX-42S, a 42 bp specific DNA with the recognition sequence for BcgI and both upstream and downstream cleavage loci; HEX-42NS, a 42 bp non-specific DNA that is identical to HEX-42S apart from single bp changes in each segment of the recognition sequence. HEX-24S and HEX-24NS are truncated versions containing the central 24 bp of HEX-42S and HEX-42NS respectively: HEX-24S thus has the recognition sequence but not the cleavage loci.
## Proteins
Transformants of Escherichia coli XL1-Blue MFR with either pARG3-BcgI or pARG3-BcgIE53A, or E. coli Rosetta 2 with pAJJ1, were grown in 20 L L-broth with the appropriate antibiotics at 37 C in a Applikon Biotechnology bioreactor to an OD 600 of 0.5-0.7. IPTG was added to 1 mM and the culture continued for 3-4 h before it was passed through a GEA Westphalia separator prior to cell harvesting by centrifugation. WT BcgI, the E53A mutant and the A protein were all purified by essentially the same procedure as described before for the WT protein [bib_ref] Concerted action at eight phosphodiester bonds by the BcgI restriction endonuclease, Marshall [/bib_ref] , except that the initial extract-after cell disruption and the removal of the debris-was fractionated, first with polyethyleneimine and then with ammonium sulphate [bib_ref] Protein precipitation techniques, Burgess [/bib_ref] , prior to the column chromatography steps. As neither BcgIE53A nor the A subunit alone possess nuclease activity, the purification of these proteins was monitored by SDS-PAGE: the final preparations of all three proteins contained no detectable impurities. The identities of the purified proteins were confirmed by N-terminal amino-acid sequencing (W. Mawby, personal communication). Approximately 50% of the A subunit carried methionine at its N-terminus, as specified by its gene sequence, and 50% valine, the second residue in the sequence: the latter presumably arises from post-synthetic processing. Protein concentrations were assessed from A 280 measurements using molar extinction coefficients calculated from amino acid compositions. Molarities of WT BcgI and E53A refer to the A 2 B assembly and that for the A protein to its monomeric state. Molecular masses encompass four possible states of the A protein [bib_ref] Organisation of the BcgI restriction-modification protein for the transfer of one methyl..., Smith [/bib_ref] : with and without the N-terminal methionine, and with or without bound SAM.
## Native mass spectrometry
Aliquots of BcgIE53A were dialysed overnight against 200 mM AmAc (ammonium acetate), 10% glycerol, pH 8.4, and flash-frozen. When required, the samples were thawed on ice and buffer-exchanged twice with 200 mM AmAc using Micro Bio-Spin P6 columns (Bio-Rad) before being diluted in AmAc to the requisite concentration. (In this buffer, E53A still bound DNA specifically and the WT enzyme cleaved DNA when MgCl 2 was added: data not shown.)
The protein sample was loaded into the mass spectrometer using nano-electrospray ionization (nano-ESI) in positive ion mode with an automated Advion Triversa inlet system (5 mm nozzle diameter, spray voltage 1.75 kV, gas pressure 0.25 psi). Mass to charge values were determined using a Waters Synapt 1 T-wave ion mobility quadrupole/time-of-flight tandem mass spectrometer with an 8 k quadrupole for medium-to-high m/z transmission. Experimental parameters were chosen to effect desolvation of high m/z sample ions while maintaining conditions that promote the transmission of intact protein complexes, with minimal disruption of native conformations (49): backing pressure, 5.4 mbar; source temperature, 25 C; sampling and extraction cone, 180 and 0 V, respectively; trap and transfer collision energies, 60 and 40 V; trap DC bias, 80 V. Data analysis was conducted using MassLynx 4.1 software (Waters) without background subtraction using smoothing by the Savitzky-Golay method (±50 channels).
## Analytical ultracentrifugation
Sedimentation equilibrium experiments were carried out at 20 C in six-channel centrepieces for a Beckman XL-A ultracentrifuge. Sample chambers contained the proteineither WT BcgI, E53A or A protein-in 110 ml analytical ultracentrifugation (AUC) buffer (20 mM Tris-HCl, pH 8.4, 66 mM NaCl, 2 mM CaCl 2 , 20 mM SAM and 10% glycerol) and reference chambers 120 ml AUC buffer. During centrifugation, absorbancies were recorded along the centrifugal radius at 230, 280 or 292 nm, depending on the protein concentration. An initial scan was made directly on reaching 3000 rpm and the horizontal trace used to determine the protein concentration in the cell. Further scans were made after !16 h at speeds between 6000 and 15 000 rpm: at least two scans, !4 h apart, were taken at each speed. A final scan was recorded after 6 h at 40 000 rpm, to yield from the meniscus a baseline absorbance (B). Data sets were fitted in ORIGIN to for the distribution of a single ideal species after sedimentation to equilibrium:
[formula] A r ¼ A r 0 Á e  ! 2 2RT M 1Àv ð Þ r 2 Àr 2 0 ð Þ Ã + Bð1Þ [/formula]
where A r and A r 0 are the absorbance at radius r and at the reference r 0 , ! the centrifugal speed, v the partial specific volume and r the solvent density: when applied to a single species, M denotes the molecular mass of that species but when applied to a mixture of species of varied mass but uniform v, M denotes the mean weight-average MW ( M w ) of all of the species in the cell. Multiple species were considered to be present whenever the residuals between the best fit to Equation 1 varied systematically from the experimental data. Further AUC experiments were carried out as above, but monitored at 539 nm, on samples containing either the HEX-42S or the HEX-42NS duplex and excess BcgI.
## Reactions
DNA cleavage rates were generally measured by adding the requisite concentration of BcgI protein to 200 ml 3 H-labelled DNA (5 nM) in buffer R (AUC buffer supplemented with 1 mM DTT and 200 mg/ml BSA, and with 10 mM MgCl 2 in place of the CaCl 2 ) at 37 C: the DNA was the SC form of either pUC19 or pDG5, which carry, respectively, one or two BcgI sites [bib_ref] Restriction endonucleases that bridge and excise two recognition sites from DNA, Marshall [/bib_ref] [bib_ref] Concerted action at eight phosphodiester bonds by the BcgI restriction endonuclease, Marshall [/bib_ref]. Samples (10 ml) were taken before and at varied times after adding the enzyme, and mixed immediately with an EDTA quench prior to electrophoresis through agarose to separate the following DNA species: the intact SC substrate; the nicked open circle (OC) form; the linear (LIN) species with at least one DSB at one site and, for the two-site substrate, the final products with at least one DSB at both sites (L1 and L2). The concentration of each form was evaluated by scintillation counting. The reactions at each BcgI concentration were repeated at least three times, although occasionally with different sampling times for each test. For repeats with the same time points, the data at each time were averaged and the averages fitted to a single exponential decay with offset. For reactions sampled at different times, the decrease in SC DNA in each reaction was fitted to an exponential and the apparent rate constants then averaged.
Reactions that were too fast to measure by manual sampling were carried out by mixing, in a Hi-Tech RQF-63 quench-flow apparatus (TgK Scientific), equal volumes (80 ml) of 3 H-labelled pDG5 (10 nM) and BcgI (at twice the required concentration), both in buffer R at 37 C. After the requisite delay time, the reaction was mixed with 80 ml EDTA (500 mM). Samples (100 ml) of the quenched reaction were then analysed by electrophoresis through agarose as above, to yield the residual concentration of the SC DNA.
For BcgI reactions in the presence of additional A protein, plasmid cleavage and the release of the 34-mer were measured in parallel [bib_ref] Concerted action at eight phosphodiester bonds by the BcgI restriction endonuclease, Marshall [/bib_ref]. The reactions, in 200 ml buffer R* (as Buffer R but 5% [w/v] Ficoll 400 instead of the glycerol) at 37 C, contained 5 nM 3 H-labelled pUC19 and specified concentrations of both native BcgI and BcgIA proteins. Samples (20 ml) were taken from the reactions at various times, mixed immediately with quench supplemented with 250 mg/ml proteinase K and incubated at 37 C for !30 min. Half of each sample was analysed by electrophoresis through agarose, as above, to evaluate the concentrations of the DNA species that could be resolved on agarose (the SC, OC and LIN forms of the plasmid). The other half was loaded onto a native 20% acrylamide gel, alongside an equivalent amount (5 nM) of HhaIdigested pUC19. The acrylamide gel was stained with SYBR Safe (Invitrogen) for 20 min, washed and its fluorescence recorded in a Typhoon PhosphorImager (Molecular Dynamics) using its 488 nm laser and 520 BP 40 emission filter. Images were analysed using ImageQuant software. The concentration of the 34-mer was determined relative to the known concentration of the 28 and 33 bp fragments from the HhaI-digested pUC19 control.
## Dna binding
Tests to measure DNA binding by BcgI were carried out at 37 C in 10 ml buffer R*C (as Buffer R* but with 2 mM CaCl 2 instead of the MgCl 2 ) containing a HEX-labelled oligoduplex (20 nM) and varied concentrations of native BcgI. After adding the protein, samples were incubated for 5 min and then loaded directly onto 6% acrylamide gels in TBC (as TBE but with 2 mM CaCl 2 in place of the EDTA) running at 4 V/cm. After loading, the voltage was increased to 12.5 V/cm. Gels were imaged in their plates in the PhosphorImager using its 532 nm laser and 555 BP 20 emission filter. Images were analysed in ImageQuant and the concentration of free DNA in each lane assessed relative to that in the lane without BcgI. The background was taken at an equivalent position in a lane lacking DNA.
# Results
Glu53 in the A subunit of the BcgI RM protein forms part of the PD. . . EXK motif common to many nuclease active sites (9) and its replacement with Ala abolishes the nuclease activity of BcgI [bib_ref] Analyzing the functional organization of a novel restriction modification system, the BcgI..., Kong [/bib_ref]. Hence, to enable studies of BcgI without its cleavage reaction, an over-producing strain for the E53A mutant of BcgIA was constructed (see 'Materials and Methods' section). Preparations of the E53A mutant from this strain yielded about twice the amount of protein as parallel preparations of the active WT enzyme. Over-expression of the WT nuclease may result in cell death due to cleavage of the chromosome at non-cognate sites, as seen with other restriction enzymes [bib_ref] Repair of the Escherichia coli chromosome after in vivo scission by the..., Heitman [/bib_ref] [bib_ref] Fidelity of DNA recognition by the EcoRV restriction/ modification system in vivo, Taylor [/bib_ref] , although the BcgI RM protein carries the MTase that ought to protect the recognition sites on the cellular DNA [bib_ref] Characterization of BcgI, a new kind of restriction-modification system, Kong [/bib_ref] [bib_ref] Analyzing the functional organization of a novel restriction modification system, the BcgI..., Kong [/bib_ref]. On the other hand, cells over-producing the inactive mutant might remain viable and so generate more of the protein. Experiments requiring high concentrations of the BcgI protein were thus facilitated by using the E53A protein rather than WT BcgI.
## Mass spectrometry
Native mass spectrometry (MS) was carried out primarily on the E53A mutant, although equivalent spectra were obtained with WT BcgI albeit with lower signal-to-noise ratios (data not shown). Under conditions where noncovalent interactions are preserved, the spectrum in [fig_ref] Figure 1: Native MS [/fig_ref] shows a series of peaks, which are assigned to the A and B subunits and their complexes with different stoichiometries. The predominant species ($75% of the total intensity) is the A 2 B complex with an m/z of around 7000, with principal charge states of 28+ and 29+. The mass calculated for the A 2 B complex is ca. 182.4 kDa, as averaged over the four different species (±N-terminal methionine and ±bound SAM; see above), which are not resolved here. The major peak in [fig_ref] Figure 1: Native MS [/fig_ref] yields an experimental mass of 182 434, which matches closely the A 2 B form of BcgI. In addition, further multiples of A 2 B assemblies were observed up to at least four such units, an (A 2 B) 4 complex at around m/z 13 000 [fig_ref] Figure 1: Native MS [/fig_ref]. The relative ratios of these higher-order complexes in the sample cannot be directly measured from the spectrum, as the sensitivity of the ion detector decreases with increasing m/z in a non-linear way. The time-of-flight system has in theory no upper limit on the size of molecule it can detect but the transmission and the detection of ions depends on their mass. It is therefore difficult to determine the relative amounts of each species in the solution.
In addition to the presence of these larger species, some separated components of the complexes were also observed. The masses of these smaller species, up to m/z 6000, correspond to the BcgIA and BcgIB subunits by themselves and to an AB assembly [fig_ref] Figure 1: Native MS [/fig_ref]. These species are likely to be subunits that had dissociated from the BcgI endonuclease in solution, as suggested by the low charge states of these species (gas-phase dissociation products would have had disproportionally high charge states, similar to denatured proteins). In an A 2 B protomer, the BcgIB subunit would normally be expected to have the weakest non-covalent linkage to the assembly as its mass, and therefore surface area, is less than that of the A subunit: this ought to make it easier for BcgIB to leave the complex than BcgIA. Hence, the presence of an AB species, but not an A 2 dimer, suggests that the two A subunits in the active BcgI protein both interact with B but not with each other: the A subunits presumably flank the B subunit, one on either side. If this suggestion is correct, then the first step in the disassembly of the protein should be the dissociation of one BcgIA subunit from the A 2 B unit to leave AB, as is seen in the spectra.
## Auc
Native MS identified several assemblies of the BcgI protein containing multiple copies of the A 2 B protomer but it does not provide quantitative information about the amount of each species. Moreover, the native MS was done in a buffer designed to permit volatilization of the sample rather than that for optimal enzyme activity. To address these issues, AUC experiments were performed on WT BcgI under conditions similar to those used for its DNA cleavage assays, though in Ca 2+ rather than Mg 2+ so as to allow for DNA binding without cleavage [bib_ref] Specific DNA recognition by EcoRV restriction endonuclease induced by calcium ions, Vipond [/bib_ref]. The inactive E53A mutant of BcgI behaved similarly to the WT in both the absence and presence of DNA and, in this case, with either Ca 2+ or Mg 2+ at hand (data not shown).
Sedimentation equilibrium experiments were carried out with varied concentrations of WT BcgI. After centrifugation for !20 h to achieve equilibrium (as evidenced by overlaying traces from successive scans !4 h apart), the radial distribution of the protein was measured by absorbance at 280 nm for protein concentrations between 1 and 10 mM; or at 230 or 292 nm for lower and higher concentrations, respectively [fig_ref] Figure 2: Sedimentation equilibrium [/fig_ref]. At each concentration, the distribution was fitted to Equation 1, for the sedimentation of a single ideal species: the fits encompassed several data sets from different times. At the lowest concentration of BcgI tested (0.4 mM), the residuals between the experimental data and the best fit to Equation 1 were both small and random, showing that the data can indeed be accounted for by a single species [fig_ref] Figure 2: Sedimentation equilibrium [/fig_ref]. The AUC data at low BcgI concentrations yielded a MW of 183 kDa, which corresponds closely to the 182.4 kDa expected for the A 2 B protomer. In contrast, the fit of the data at high concentrations to Equation 1 gave large and non-random residuals [fig_ref] Figure 2: Sedimentation equilibrium [/fig_ref] , suggesting either the presence of multiple species and/or non-ideality. In this situation, the best fit to Equation 1 does not yield the MW of any individual species but rather the mean weightaverage MW (M w ) of all of the species in the sample.
Values for M w were obtained at each loading concentration of BcgI tested . The M w values increased with increasing levels of BcgI: from that corresponding to a single A 2 B unit at low concentrations, then to values consistent with dimers of A 2 B and finally to levels indicating assemblies with more than two protomers. At the highest concentration tested (18 mM: [fig_ref] Figure 2: Sedimentation equilibrium [/fig_ref] , the M w came to 431 kDa. As these mean values for M w encompass the full range of protein concentrations present in the centrifuge cell after sedimentation to equilibrium, from low concentrations at the inside edge to high at the outside, a significant fraction of the protein in this respectively. The plots above the main panels display the residuals between these fits and the experimental data: the random variations in (a) show that the record can be accounted for by a single ideal species with a unique value of M; the systematic deviations in (b) demonstrate that more than one species is present, which gives rise to a M w value. . Self-association of BcgI. Varied concentrations of BcgI in AUC buffer were loaded into the AUC and sedimented to equilibrium at 7500 rpm at 20 C. For each sample, the absorbance was measured as a function of centrifugal radius and fitted to Equation 1. Apart from the data at the lowest BcgI concentrations (viz. [fig_ref] Figure 2: Sedimentation equilibrium [/fig_ref] , the residuals between the fits and the experimental data displayed non-random deviations (viz. [fig_ref] Figure 2: Sedimentation equilibrium [/fig_ref] sample must be present in assemblies containing more than two protomers.
AUC was also used to analyse complexes of WT BcgI with either a specific duplex carrying both recognition and cleavage sites (HEX-42S) or a non-specific DNA that lacked the recognition sequence (HEX-42NS). Sedimentation was monitored at 539 nm, to detect the HEX moiety on the DNA, either in its free state or when enzyme-bound. In the absence of BcgI, MW values of 27.6 and 26.6 kDa were obtained for HEX-42S and HEX-42NS, respectively (data not shown), close or equal to the expected MW, 26.6 kDa. When the non-specific duplex (2 mM) was centrifuged in the presence of excess BcgI (7.5 mM), the initial uniform distribution of the HEX label throughout the cell, black trace) was converted into the characteristic profiles for an approach to sedimentation equilibrium, coloured traces). The records at equilibrium (>22 h) yielded a M w of 349 kDa, much larger than that for the free DNA so all of the DNA must be bound to the protein. The M w for the non-specific complex is similar to that for the same concentration of free protein , which indicates that either two or more protomers can bind to one duplex or that the DNAprotein complexes associate with themselves. Conversely, after adding BcgI (7.5 mM) to the specific duplex (2 mM) and spinning the sample at low velocity, the solution lacked virtually any absorbance at 539 nm, black trace). Subsequent scans after centrifugation at a higher speed revealed a progressive recovery in the A 539 signal, coloured traces) but even after 54 h, the total absorbance in the cell containing specific DNA was still lower than that containing non-specific DNA and had yet to reach equilibrium. The complex of BcgI with specific DNA must precipitate out of solution as soon as it is formed and then slowly redissolves back into solution. An 18 bp duplex that contains the recognition site but not the cleavage loci also precipitated from solution on the addition of WT BcgI, as did the specific 42 bp duplex with E53A in the presence of either Ca 2+ or Mg 2+ (data not shown).
## Dna cleavage
During its DNA cleavage reaction, the BcgI protein interacts with two copies of its recognition sequence and then proceeds to make four DSBs, one either side of each site, thus cutting a total of eight phosphodiester bonds in a single DNA-protein complex [bib_ref] Concerted action at eight phosphodiester bonds by the BcgI restriction endonuclease, Marshall [/bib_ref]. It is highly unlikely that one A 2 B protomer can cut all eight bonds because it contains only two active sites for phosphodiester hydrolysis, one in each A subunit. Instead, it seems more likely that DNA cleavage is carried out by an assembly of A 2 B units, maybe like those seen by native MS and AUC. This possibility was addressed by analysing how the extent of cleavage of plasmids with one or two BcgI sites varied as the protein concentration was raised from below to above the level of BcgI sites in the DNA . BcgI can act with two recognition sites in cis or in trans, although, as is usual for interactions bridging two loci [bib_ref] Enzyme-mediated DNA-looping, Halford [/bib_ref] , it prefers sites in cis over those in trans. Two-site substrates are thus cleaved more readily than one-site DNA [bib_ref] Restriction endonucleases that bridge and excise two recognition sites from DNA, Marshall [/bib_ref] [bib_ref] Does BcgI, a unique restriction endonuclease, require two recognition sites for cleavage?, Kong [/bib_ref].
In previous studies [bib_ref] Does BcgI, a unique restriction endonuclease, require two recognition sites for cleavage?, Kong [/bib_ref] , the extent of cleavage of plasmids with one or two BcgI sites was measured at varied BcgI concentrations after a fixed time (15 min): complete cleavage of the DNA within this time required a molar excess of protein over DNA, with more protein needed for the one-site than for the two-site DNA. As the extent of cleavage after a fixed time interval may reflect either the end point or the rate of the reaction, reactions at varied BcgI concentrations were monitored in this study over extended time bases . On the one-site plasmid, no-or little-cleavage was detected at BcgI levels below or equal to that of the DNA, even after prolonged incubation . Instead, the reaction on the one-site substrate required enzyme concentrations in excess of the DNA. The extent of cleavage increased as the ratio of enzyme to recognition sites was raised above 1:1 but complete cleavage was only observed with a substantial excess of protein over DNA. The plasmid with . AUC of BcgI-DNA complexes. The samples, in AUC buffer at 20 C, contained WT BcgI protein (7.5 mM) and a DNA duplex (2 mM) as follows: (a) HEX-42NS, a non-specific DNA that lacks the recognition sequence; (b) HEX-42S, a specific DNA with both recognition and cleavage loci. The samples were centrifuged in the AUC and the absorbance across the cell monitored at 539 nm at the following stages after initiating the run: the instrument was initially set to spin at 3000 rpm and an absorbance scan recorded immediately on reaching that speed (black trace). The velocity was then raised to 7500 rpm and scans taken after 14 h and after further 8 h delays. The records shown are after 14 h (green trace), 22 h (cyan trace), 38 h (pink trace) and 54 h (blue trace). The data sets with the non-specific duplex (a) recorded after !30 h were fitted to Equation 1: the best fit gave a M w of 349 kDa. two cognate sites might have been expected to require more enzyme per mole DNA to fill both sites but instead almost all of the two-site DNA was cleaved at equal levels of enzyme and DNA: i.e. with BcgI at half the concentration of recognition sites .
Enzyme concentrations that gave complete cleavage of the DNA did not necessarily give maximal reaction rates: for instance, the two-site plasmid (present at 5 nM) was cleaved fully by both 10 and by 20 nM BcgI protein but the latter gave a faster rate . Cleavage of both one-and two-site substrates was therefore studied across a range of BcgI concentrations far above that of the DNA . During these reactions, the concentrations of the SC DNA substrates fell exponentially to end points where essentially all of the DNA had been cleaved. With enzyme in excess over the DNA, the rate constants (k obs ) from the exponential decline of the SC DNA denote the cleavage of at least one phosphodiester bond in the enzyme-DNA complex rather than the entire reaction ending with the release of the DNA cleaved at eight bonds. Phosphodiester hydrolysis by BcgI bound to the one-site substrate took !2 min to reach completion and could be monitored by manually mixing the reagents . In contrast, BcgI bound to the two-site plasmid could take <0.2 min to cut all of the DNA, so in these cases the experiments were done in a rapid quench-flow device . At BcgI concentrations that permitted both hand-mixing and quench-flow approaches, the two techniques yielded comparable results (data not shown).
The values for k obs : other data not shown) were plotted against the enzyme concentration . For both one-and two-site plasmids, the rates increased with increasing BcgI concentrations in a hyperbolic manner . The data with each substrate were fitted to an equation for a rectangular hyperbola to yield values for k max (the rate constant for DNA cleavage at saturation of the DNA with protein) and for . DNA cleavage at low . Reactions in buffer R at 37 C contained one of the following plasmids ( 3 H-labelled, initially $90% SC monomer) at 5 nM and BcgI protein at the concentrations noted below. The plasmids were (a) pUC19, which has one BcgI site; (b) pDG5, which has two sites. The enzyme concentrations for each reaction are marked on the right: 2 nM, white circles; 5 nM, black circles; 10 nM, white squares; 20 nM, black squares. Samples were taken at the indicated times and analysed by electrophoresis to separate the SC substrate from the cleaved products. The residual concentrations of the SC DNA were determined and are plotted as a function of time. The lines are the best fits to single exponential decays with floating offsets. . DNA cleavage at high . (a) The reactions, in buffer R at 37 C, contained 5 nM 3 H-labelled SC pUC19 and BcgI protein at 75 nM, white circles; 250 nM, black circles; 750 nM, white squares. After manually mixing the reagents, samples were taken from the reactions by pipette and mixed immediately with an EDTA quench. The samples were then subjected to electrophoresis through agarose, to enable the concentration of SC DNA to be determined. The amount of SC DNA present in each sample is plotted against the time. (b) As (a) except that the DNA was SC pDG5 and that the reactions were carried out by quench-flow. (c) Apparent rate constants (k obs ) from !3 independent experiments at each BcgI concentration were averaged and plotted against the concentration: for reactions on pUC19, white circles; for reactions on pDG5, black circles. Error bars denote standard deviations. The line drawn through each data set is the best fit to a hyperbolic function, k obs = (k max  [BcgI])/(K ½ +[BcgI]). The best fits were obtained with the following: for pUC19, k max = 2.7 min À1 and K ½ = 232 nM; for pDG5, k max = 21 min À1 and K ½ = 230 nM.
K ½ (the protein concentration for half maximal rate). The two-site plasmid gave nearly a 10-fold higher value for k max than the one-site DNA, 21 min À1 compared with 2.7 min À1 . In contrast, the one-and two-site substrates gave the same value for K ½ , 230 nM in both cases. The faster rate of cutting the two-site over the one-site plasmid is thus due solely to the different k max values. The DNA cleavage step in the BcgI reaction on sites in cis is thus faster than that on sites in trans.
The values for K ½ merit two comments. Firstly, they indicate that remarkably high BcgI concentrations are required for its maximal reaction rate: cleavage of 5 nM DNA at 90% of its maximal rate requires an enzyme concentration of >2000 nM. Secondly, though proteins that bind two DNA sites nearly always have higher affinities for sites in cis than sites in trans (16), the equal K ½ values show that reactions with two sites in trans feature the same affinity for BcgI protein as that during reactions with sites in cis. The K ½ values may therefore reflect a weak proteinprotein association in the assembly of the reaction complex rather than the DNA-protein association.
## Dna binding
An alternative explanation for why DNA cleavage by BcgI requires such large amounts of protein is that the enzyme simply has a low affinity for its recognition sequence. To test this possibility, the binding of the BcgI protein to specific and non-specific DNA was examined directly by gel retardation [fig_ref] Figure 7: DNA binding [/fig_ref]. The binding studies were initially done with two 42 bp oligoduplexes, both labelled with HEX at one 5 0 terminus. One, HEX-42S, is a potential substrate, as it carries both the recognition sequence and the cleavage loci both sides of the site. The other, HEX-42NS, differs only at 2 bp, one in each segment of the bipartite site so is effectively a non-specific sequence for BcgI [bib_ref] Concerted action at eight phosphodiester bonds by the BcgI restriction endonuclease, Marshall [/bib_ref]. The buffer used here contained Ca 2+ rather than Mg 2+ , to allow WT BcgI to bind to HEX-42S without cutting off its ends. Increasing concentrations of BcgI were added to a fixed amount of each DNA and the free DNA then separated by electrophoresis from the protein-bound species with retarded mobilities [fig_ref] Figure 7: DNA binding [/fig_ref]. The extent of binding at each enzyme concentration was then determined by assessing the fluorescence from the band corresponding to the free DNA relative to that in samples without enzyme [fig_ref] Figure 7: DNA binding [/fig_ref]. Similar results (not shown) were obtained with the inactive E53A mutant with either Ca 2+ or Mg 2+ and with no metal present.
When increasing concentrations of BcgI were added to the specific duplex, HEX-42S, the concentration of the free DNA decreased linearly with the protein concentration until it had all been converted to DNA-protein complexes [fig_ref] Figure 7: DNA binding [/fig_ref]. BcgI thus binds to DNA carrying its recognition sequence in a stoichiometric fashion, where all of the added BcgI is bound to the DNA until saturation of the DNA is achieved, as opposed to a dynamic equilibrium between free and bound proteins. However, saturation of the specific duplex seems to require 3-4 mole BcgI per mole DNA, as it took $70 nM BcgI to saturate 20 nM DNA [fig_ref] Figure 7: DNA binding [/fig_ref] , intersection of red lines). BcgI also bound to the non-specific DNA, HEX-42NS, although with a lower affinity than the specific sequence [fig_ref] Figure 7: DNA binding [/fig_ref]. Moreover, the specific and non-specific sequences gave rise to different complexes. The specific DNA was initially transformed to species that failed to enter the polyacrylamide gel and remained in the wells [fig_ref] Figure 7: DNA binding [/fig_ref] : species that entered the gel were observed only after sufficient enzyme had been added to convert all of the DNA to complexes. The nature of the complexes that remain in the wells and those that enter the gel have yet to be established. They may be related to the complexes seen in the AUC, where the addition of BcgI to cognate DNA led initially to an insoluble complex which then slowly redissolved back into solution. In contrast, all of the complexes formed with the non-specific DNA entered the gel [fig_ref] Figure 7: DNA binding [/fig_ref] but, as with the specific DNA, the species within the gel displayed progressively reduced mobilities with increasing amounts of BcgI, probably due to the binding of additional molecules of protein to each molecule of DNA.
Further experiments were carried out with 24 bp duplexes with or without the recognition sequence, HEX-24S and HEX-24NS: the former contains the recognition sequence but lacks the cleavage loci 10 and 12 nt away from the site. Nevertheless, BcgI bound to both of these 24 bp duplexes in the same manners (gels not shown) and with the same affinities [fig_ref] Figure 7: DNA binding [/fig_ref] as their 42 bp counterparts. The cleavage loci are therefore not needed for specific binding to the site.
A striking feature of the equilibrium binding of BcgI to its cognate sequence is that a much lower concentration of the protein is required to saturate binding to the site [fig_ref] Figure 7: DNA binding [/fig_ref] compared with the amount needed to saturate the DNA cleavage rate : 70 cf. >2000 nM. A scheme that can account for this behaviour is a mechanism of the type seen with the FokI restriction enzyme [bib_ref] FokI dimerization is required for DNA cleavage, Bitinaite [/bib_ref] [bib_ref] Protein assembly and DNA looping by the FokI restriction endonuclease, Catto [/bib_ref] [bib_ref] Illuminating the reaction pathway of the FokI restriction endonuclease by fluorescence resonance..., Pernstich [/bib_ref] ,
[formula] E+E+S K DNA Ð E:S+E K prot Ð E 2 S kmax À! E 2 P [/formula]
In this scheme, one molecule of the enzyme (E) first binds to its DNA site (S) with an equilibrium dissociation constant K DNA but the resultant E.S complex has no activity until the DNA-bound protein associates with a second protein, with a constant K prot for the proteinprotein assembly: the complex containing two molecules of enzyme then proceeds to cleave the DNA at the k max rate. In the case of FokI [bib_ref] Protein assembly and DNA looping by the FokI restriction endonuclease, Catto [/bib_ref] [bib_ref] Illuminating the reaction pathway of the FokI restriction endonuclease by fluorescence resonance..., Pernstich [/bib_ref] , the K D for the binding of the enzyme to its site (4 nM) is much smaller than that for the protein-protein association (in the range from 100 to 166 nM), so the level of FokI needed for its maximal DNA cleavage rate is much higher than that required to fill its binding sites on the DNA. The same seems to apply to BcgI, though the stoichiometry of the binding reaction [fig_ref] Figure 7: DNA binding [/fig_ref] implies that even its initial E.S complex must contain more than one A 2 B unit, and that it then proceeds to bind yet more A 2 B units. Indeed, if the K ½ from its maximal cleavage rate (230 nM) corresponds to its value for K prot , then the E.S complexes for BcgI and for FokI display similar affinities for their additional enzyme.
## Activation by bcgia
The monomer of FokI at its recognition site can be activated by associating not only with intact FokI protein but also with just its catalytic domain [bib_ref] FokI dimerization is required for DNA cleavage, Bitinaite [/bib_ref]. The association of the intact protein and the catalytic domain generates a unit with two active sites that can then proceed to cut both DNA strands [bib_ref] Structure of FokI has implications for DNA cleavage, Wah [/bib_ref]. The nuclease function of the BcgI protein resides in its A subunit while the sole function of B is DNA recognition [bib_ref] Characterization of BcgI, a new kind of restriction-modification system, Kong [/bib_ref] [bib_ref] Analyzing the functional organization of a novel restriction modification system, the BcgI..., Kong [/bib_ref]. Hence, it might be possible to activate the BcgI REase at its recognition site by excess A protein, isolated from the B subunit. To test this idea, the bcgIA gene was cloned and over-expressed in E. coli cells that lacked bcgIB and the A protein purified to homogeneity (see 'Materials and Methods' section). In solution, BcgIA was found by AUC and native MS to exist primarily as a 71.5 kDa monomer. The fact that the isolated BcgIA protein exists as a monomer concurs with the native MS on the native BcgI protein, which had shown A monomers, AB and A 2 B assemblies, but no A 2 dimers [fig_ref] Figure 1: Native MS [/fig_ref].
When the purified A protein was added by itself to the one-site plasmid pUC19, no DNA cleavage was observed even after long incubations with high BcgIA concentrations [fig_ref] Figure 8: Activation of BcgI by BcgIA [/fig_ref]. BcgIA was then added to pUC19 together with intact BcgI protein. However, the latter was present at the same concentration (5 nM) as the DNA, which is too low for it to cleave the DNA unaided . Samples were taken from the mixture and a portion of each analysed by electrophoresis through agarose to separate the SC, OC and LIN forms of pUC19. Though neither protein alone was capable of cutting this DNA, together they rapidly converted most of the SC plasmid directly to LIN DNA, without accumulating nicked OC forms en route [fig_ref] Figure 8: Activation of BcgI by BcgIA [/fig_ref]. The LIN DNA could have been cut in both strands on just one side of the BcgI site, to give a LIN species of 2686 bp, or on both sides to release the 34-mer product and a LIN DNA of 2652 bp. As these LIN forms are inseparable on agarose, a second portion from each sample was applied to a polyacrylamide gel to capture the 34-mer [bib_ref] Concerted action at eight phosphodiester bonds by the BcgI restriction endonuclease, Marshall [/bib_ref]. The increase in the concentration of the 34-mer matched that for the LIN DNA [fig_ref] Figure 8: Activation of BcgI by BcgIA [/fig_ref]. The A protein must therefore enable the intact protein to make DSBs on both sides of its site. Hence, both A subunits in the A 2 B protomer at the recognition site interact with free BcgIA monomers to give an assembly with two pairs of nuclease active sites, each of which makes a DSB. If the addition of A to A 2 B had resulted in only one unit for DSBs, it would have first cut one side of the site and only later the other side: the 34-mer would then have been liberated not at the same time as the LIN species but instead at some later time. Strikingly, extra A subunits also enhance the MTase activity of the BcgI protein [bib_ref] Organisation of the BcgI restriction-modification protein for the transfer of one methyl..., Smith [/bib_ref] but, as the MTase methylates only one adenine at a time, the activation of the MTase almost certainly does not require the catalytic functions for methyl transfer in the free A subunit. In contrast, the liberation of the 34-mer shows that the extra A subunits contribute to the BcgI REase functional active sites for phosphodiester hydrolysis.
The effect of the A protein was also examined at higher concentrations of native BcgI to see whether or not additional BcgIA could still enhance the cleavage rate. In reactions containing 25 nM WT BcgI and 5 nM pUC19, all of the DNA was cleaved readily . When these reactions were supplemented with 100 nM BcgIA (as in [fig_ref] Figure 8: Activation of BcgI by BcgIA [/fig_ref] , no difference was observed in either the extent or the rate of the reaction relative to that in the absence of the A protein (data not shown). Hence, the additional catalytic domains that the REase at the recognition site needs for its nuclease reaction can come from either free A subunits or excess A 2 B protomers. Nevertheless, the activation requires a lower concentration of A 2 B units than free A subunits.
# Discussion
The substrate for the REase activity of the BcgI RM protein [fig_ref] Figure 9: Scheme for action at eight phosphodiester bonds by BcgI [/fig_ref] consists of two copies of its recognition sequence [bib_ref] Restriction endonucleases that bridge and excise two recognition sites from DNA, Marshall [/bib_ref] [bib_ref] Does BcgI, a unique restriction endonuclease, require two recognition sites for cleavage?, Kong [/bib_ref] [bib_ref] Concerted action at eight phosphodiester bonds by the BcgI restriction endonuclease, Marshall [/bib_ref]. In contrast to the many restriction enzymes that are most active when bound to two sites but which still have some activity when bound to a single site [bib_ref] DNA synapsis through transient tetramerization triggers cleavage by Ecl18kI restriction enzyme, Zaremba [/bib_ref] [bib_ref] Conversion of the tetrameric restriction endonuclease Bse634I into a dimer: Oligomeric structure-stability-function..., Zaremba [/bib_ref] [bib_ref] A switch in the mechanism of communication between the two DNA-binding sites..., Bellamy [/bib_ref] [bib_ref] DNA looping by FokI: the impact of twisting and bending rigidity on..., Laurens [/bib_ref] , the BcgI REase has an absolute requirement for two sites. It has no detectable activity when bound to a single site on a DNA held in physical isolation from any other DNA, but that isolated site is cleaved when a second specific DNA is added [bib_ref] Concerted action at eight phosphodiester bonds by the BcgI restriction endonuclease, Marshall [/bib_ref]. At each site, it cuts both strands at two separate loci, one 10/12 nt upstream and the other 12/10 nt downstream [bib_ref] A unique restriction endonuclease, BcgI, from Bacillus coagulans, Kong [/bib_ref]. After binding to its substrate, the cleavage reaction yields none of the nicked species cut in one strand and only low levels of the intermediates with one DSB. Instead, most of the DNA is converted directly to the final products with two DSBs at both sites. Hence, after spanning two sites, the REase can cut all eight bonds within the lifetime of the synaptic complex [bib_ref] Concerted action at eight phosphodiester bonds by the BcgI restriction endonuclease, Marshall [/bib_ref]. To achieve this feat, the REase activity of BcgI most likely requires an assembly containing eight catalytic centres for phosphodiester hydrolysis. Given the highly concerted nature of its reaction, it is unlikely that it sequentially moves its catalytic centres from bond to bond, especially not from a bond in the 5 0 -3 0 strand to one in the 3 0 -5 0 strand.
The A subunit of the BcgI RM system carries both REase and MTase functions, while B resembles a DNA specificity subunit (HsdS) from a Type I RM system [bib_ref] Characterization of BcgI, a new kind of restriction-modification system, Kong [/bib_ref] [bib_ref] Analyzing the functional organization of a novel restriction modification system, the BcgI..., Kong [/bib_ref]. HsdS subunits recognize bipartite sequences via separate domains for each segment, with each domain linked to a conserved region that associates with a MTase subunit in a pseudo-symmetric fashion [bib_ref] A symmetrical model for the domain structure of Type I DNA methyltransferases, Kneale [/bib_ref]. BcgIB appears to be organized similarly [bib_ref] Analyzing the functional organization of a novel restriction modification system, the BcgI..., Kong [/bib_ref]. Following SDS-PAGE, it appeared from protein staining that BcgI contained two mole A per mole B (K94), but gel stains are often far from quantitative [bib_ref] Quantitative proteomics: assessing the spectrum of in-gel protein detection methods, Gauci [/bib_ref]. Nevertheless, native MS of the intact protein under conditions that minimize the disruption of non-covalent complexes (49) validated the earlier proposal and showed that BcgI is indeed composed of A 2 B units [fig_ref] Figure 1: Native MS [/fig_ref]. Under these conditions, however, we also find some lone A and B subunits and an AB assembly but no A 2 dimers. In all likelihood, the B subunit occupies the central position in the A 2 B assembly with A subunits symmetrically disposed on either side, both interacting with B but not with each other. This configuration is the same as that for the M 2 S structure of a Type I MTase (6,57) and a similar arrangement has been proposed for the AhdI MTase, a system that has a Type II REase but a Type I-like MTase [bib_ref] Shape and subunit organisation of the DNA methyltransferase M.AhdI by small-angle neutron..., Callow [/bib_ref]. Native MS also revealed the presence of species containing multiple copies of the A 2 B protomer, from an (A 2 B) 2 dimer to at least up to a (A 2 B) 4 tetramer, albeit with progressively decreasing peak heights at higher masses [fig_ref] Figure 1: Native MS [/fig_ref]. Yet, while native MS can identify these larger species, peak heights do not provide quantitative information about the amount of each species present in the sample as it was injected from the nano-ESI, since the sensitivity of the ion detector declines with increasing m/z values. Nonetheless the spectra indicated no preference for one oligomer over another, as the intensity of the peaks from the aggregates declined steadily with size. AUC was therefore used to investigate the selfassociation of the BcgI protein [fig_ref] Figure 2: Sedimentation equilibrium [/fig_ref]. At sub-micromolar concentrations, BcgI existed in solution as a single ideal species with a MW matching that for an individual protomer [fig_ref] Figure 2: Sedimentation equilibrium [/fig_ref]. But at micomolar concentrations, of the order of those required for maximal nuclease activity , multiple aggregates were present [fig_ref] Figure 2: Sedimentation equilibrium [/fig_ref]. The M w values increased with increasing concentrations of BcgI, to levels above that for a dimeric (A 2 B) 2 unit . As the M w values reflect all of the species in the cell, the mixtures must contain some of greater mass than the dimer. BcgI thus readily associates with itself to give large assemblies containing several copies of the A 2 B protomer. Its complex with non-specific DNA also contained multiple protomers. In contrast, the addition of BcgI to a specific duplex gave an insoluble complex that precipitated immediately from solution, as judged by its removal from solution on centrifugation at low velocityand by its inability to enter a polyacrylamide gel on electrophoresis [fig_ref] Figure 7: DNA binding [/fig_ref]. The binding of BcgI to specific DNA thus generates highly aggregated states that possibly link together several DNA molecules, but these then slowly dissociate to release individual DNA-protein complexes back into solution.
The cleavage of a plasmid with one BcgI site required excess protein over DNA , which raises the possibility that this REase may carry out just one turnover. It had been suggested that further cleavage reactions were blocked by the excised 34-mer remaining bound to the enzyme while it was being methylated [bib_ref] Characterization of BcgI, a new kind of restriction-modification system, Kong [/bib_ref].
Yet the fact that nuclease reactions on a plasmid with two sites proceed to completion when the enzyme is present at lower concentrations than sites on the DNA shows that the BcgI REase is capable of multiple turnovers, with each enzyme molecule participating in more than one cleavage event. BcgI is therefore not a suicide enzyme [bib_ref] Suicide inactivation of the E. coli O6-methylguanine-DNA methyltransferase, Lindahl [/bib_ref] [bib_ref] Suicide substrates, mechanism-based enzyme inactivators: recent developments, Walsh [/bib_ref] , one that is inactivated by its own reaction product. The need for excess enzyme for its reaction in trans on one-site DNA, despite its reactions in cis featuring multiple turnovers, can be accounted for if the BcgI proteins bound to both recognition sites have to recruit additional molecules of protein before they become active. In this situation, a reaction in trans will require two DNA molecules to be both loaded with enzyme at the recognition site and with the extra enzyme. But if the enzyme binds tightly to its cognate site as seems to be the case here [fig_ref] Figure 7: DNA binding [/fig_ref] , fully loaded molecules of the one-site DNA carrying the extra enzyme will appear in solution only after adding a stoichiometric excess of enzyme over sites. Conversely, for a reaction in cis, the BcgI molecules bound to the two recognition sites in the same DNA molecule may interact with each other cooperatively [bib_ref] Enzyme-mediated DNA-looping, Halford [/bib_ref] , which could lead directly to the capture of the additional molecules of protein required for the cleavage reaction.
When bound to its recognition site, the A 2 B protomer is in a position to place its two nuclease domains against two of the four target phosphodiester bonds at that site [fig_ref] Figure 9: Scheme for action at eight phosphodiester bonds by BcgI [/fig_ref]. This would give a structure similar to that for M.AhdI [bib_ref] Shape and subunit organisation of the DNA methyltransferase M.AhdI by small-angle neutron..., Callow [/bib_ref] , an M 2 S MTase but which is thought from neutron scattering data to have arms extending from both M subunits. In the scheme shown in [fig_ref] Figure 9: Scheme for action at eight phosphodiester bonds by BcgI [/fig_ref] , the nuclease domains of the A subunits are placed against the À10 and +10 targets in the two strands, one upstream and one downstream of the site, but an equally plausible model would put these domains against the À12 and +12 bonds. On the other hand, it is improbable that an A 2 B unit places one nuclease centre against a bond 10 nt distant from the site and the other 12 nt away, as this would break the intrinsic symmetry of the system. Moreover, the positioning of one nuclease domain at a 10 position and the other at a 12 position calls for an implausible reconfiguration of one A subunit: an attack on both scissile bonds in the same strand (say À10 and +12) requires a 180 rotation of the nuclease domain in one A subunit relative to the other, to keep register with strand polarity; alternatively, to attack both strands at one cleavage locus (say À10 and À12), the distance between the nuclease and MTase domains in one A subunit would have to be massively different from that in the other. The synaptic complex of two A 2 B protomers and two recognition sites [fig_ref] Figure 9: Scheme for action at eight phosphodiester bonds by BcgI [/fig_ref] would thus seem to be incapable of making a DSB at any single cleavage locus. Indeed, it is probably incapable of cutting even one bond since no nicked species appear during DNA cleavage by BcgI [fig_ref] Figure 1: Native MS [/fig_ref].
The monomer of the FokI REase binds to its asymmetric site via its DNA recognition domain [bib_ref] Structure of the multimodular endonuclease FokI bound to DNA, Wah [/bib_ref] and then moves its catalytic domain to its target bond, 13 nt away in the bottom strand, but the REase remains inactive, not even nicking the DNA [bib_ref] Illuminating the reaction pathway of the FokI restriction endonuclease by fluorescence resonance..., Pernstich [/bib_ref]. It becomes active only after a second catalytic centre engages the target bond in the top strand. The second centre can come from another monomer of the FokI protein, either in free solution or bound elsewhere to the same DNA (31), or from just its catalytic domain [bib_ref] FokI dimerization is required for DNA cleavage, Bitinaite [/bib_ref]. A similar scheme may also apply to the BcgI REase [fig_ref] Figure 9: Scheme for action at eight phosphodiester bonds by BcgI [/fig_ref]. Much higher concentrations of BcgI protein are required to cleave DNA than to bind to specific DNA [fig_ref] Figure 7: DNA binding [/fig_ref] so the complex formed at the cognate site must remain inactive until it recruits additional molecules of the protein from free solution. BcgI bound to its site can also be activated by the A subunit alone [fig_ref] Figure 8: Activation of BcgI by BcgIA [/fig_ref]. The function of either the additional A 2 B protein, or BcgIA alone, is presumably to associate transiently with the A subunits of the DNA-bound enzyme to position two catalytic centres for phosphodiester hydrolysis at each cleavage locus: the two centres can then cut both strands at that locus. Moreover, as the activation of the BcgI REase by the A protein alone leads directly to the excision of the 34-mer [fig_ref] Figure 8: Activation of BcgI by BcgIA [/fig_ref] , both A subunits in the A 2 B protomer on the DNA must interact with free A protein before any reaction. [fig_ref] Figure 9: Scheme for action at eight phosphodiester bonds by BcgI [/fig_ref] shows a proposal for the juxtaposition of eight catalytic centres against the eight target bonds in the BcgI substrate. In this scheme, one A 2 B protomer binds to each recognition site and positions its two centres for nuclease activity, one in each A subunit, against one particular set of phosphodiester bonds, illustrated here [fig_ref] Figure 9: Scheme for action at eight phosphodiester bonds by BcgI [/fig_ref] as the À10 and +10 targets. Two additional A 2 B protomers from free solution then become incorporated into the synaptic complex through interactions between their nuclease domains and the nuclease domains of the DNA-bound proteins [fig_ref] Figure 9: Scheme for action at eight phosphodiester bonds by BcgI [/fig_ref]. The additional protomers bridge the two DNA sites, so that (in the configuration drawn) one of the extra protomers engages the À12 targets upstream of both sites and the other the +12 targets downstream of both sites. Alternative configurations for the bridging protomers are also possible: for example, they could span ''diagonally'' the upstream locus at one site and the downstream locus at the other site. Indeed, the insolubility of the specific DNA-protein complex for BcgI seen in the AUCcould be due to the two bridging protomers spanning not the same two DNA-bound protomers to form the closed structure shown in [fig_ref] Figure 9: Scheme for action at eight phosphodiester bonds by BcgI [/fig_ref] but rather an open-ended structure with one DNA-bound protomer being linked via bridging units to two different DNA-protein complexes.
[fig] Figure 1: Native MS. A nano-ESI mass spectrum of the E53A form of BcgI(27.5 mM) in 200 mM AmAc was recorded under non-denaturing conditions (see 'Materials and Methods' section). The insert shows the signals for the oligomeric states in the m/z range from 8500 to 14 500 magnified 5-fold. The charge state series assigned to each molecular species (in bold) are indicated with dashed brackets, and the charge and m/z value for the primary peak in each series is given. [/fig]
[fig] Figure 2: Sedimentation equilibrium. Samples of BcgI in AUC buffer were centrifuged at 7500 rpm at 20 C for the times noted below. The main panels show the absorbance (white circles) as a function of the centrifugal radius: (a) 0.44 mM BcgI at 230 nm after 30 h; (b) 18 mM BcgI at 292 nm after 44 h. The lines through the data points in the main panels are the best fits of the following data sets to Equation 1: (a) the records after 26 and 30 h; (b) after 32, 38 and 44 h. The best fits were obtained with values for M of 183 and 431 kDa in (a) and (b) [/fig]
[fig] Figure 4: AUC of BcgI-DNA complexes. The samples, in AUC buffer at 20 C, contained WT BcgI protein (7.5 mM) and a DNA duplex (2 mM) as follows: (a) HEX-42NS, a non-specific DNA that lacks the recognition sequence; (b) HEX-42S, a specific DNA with both recognition and cleavage loci. The samples were centrifuged in the AUC and the absorbance across the cell monitored at 539 nm at the following stages after initiating the run: the instrument was initially set to spin at 3000 rpm and an absorbance scan recorded immediately on reaching that speed (black trace). The velocity was then raised to 7500 rpm and scans taken after 14 h and after further 8 h delays. The records shown are after 14 h (green trace), 22 h (cyan trace), 38 h (pink trace) and 54 h (blue trace). The data sets with the non-specific duplex (a) recorded after !30 h were fitted to Equation 1: the best fit gave a M w of 349 kDa. [/fig]
[fig] Figure 7: DNA binding. (a and b) The mixtures contained, in 10 ml buffer R*C, 20 nM DNA, either HEX-42S (a) or HEX-42NS (b), and BcgI at one of the following levels, from left to right: 0, 20, 40, 60, 80, 100, 150 or 200 nM. After 5 min at 37 C, the samples were subjected to electrophoresis through polyacrylamide and the fluorescence from each band recorded. On the left of gel images, F marks the position of the free DNA; C, the DNA-protein complexes that entered the gel; and W, the bottom of the wells. (c) From titrations of both HEX-42S and HEX-42NS with increasing amounts of BcgI protein, and from additional experiments with 24 bp duplexes (HEX-24S and HEX-24NS: gels not shown), the amount of free DNA in each sample was determined relative to the amount in a parallel lane without enzyme. The % DNA in the free form is shown as a function of the BcgI concentration: HEX-42S, white triangle; HEX-42NS, white squares; HEX-24S, black triangles; HEX-24NS, black squares. Mean values from three repeats are given: error bars show standard deviations. The red lines through the data with the specific duplexes denote stoichiometric binding where all of the added BcgI is bound to DNA until saturation of the DNA is achieved. [/fig]
[fig] Figure 8: Activation of BcgI by BcgIA. The reactions, in 200 ml buffer R* at 37 C, contained 100 nM BcgIA protein and 5 nM 3 H-labelled pUC19 (initially 90% SC monomer), in either the absence or presence of 5 nM WT BcgI. Aliquots (20 ml) were removed at the times shown and immediately quenched before analysis by electrophoresis: one-half through agarose, to separate the SC, OC and LIN forms of the plasmid; the other half through polyacrylamide, to capture the 34-mer. The concentrations of the various forms of pUC19 were evaluated from the agarose gel and that for the 34-mer from the polyacrylamide gel (see 'Materials and Methods' section). For the reaction with BcgIA alone, only the level of SC DNA is shown: red unfilled triangles and line. For the reaction with both BcgIA and native BcgI, the following were assessed: intact SC DNA, white circles; nicked OC DNA, white squares; LIN DNA, with at least one DSB at the BcgI site, white inverted triangles; the 34-mer, black triangles. Mean values from three repeats are shown: error bars denote standard deviations. [/fig]
[fig] Figure 9: Scheme for action at eight phosphodiester bonds by BcgI. (a) The substrate for the REase activity of BcgI consists of two copies of its recognition sequence. The sites, shown here aligned in parallel, can be located either on the same DNA molecule, in cis, or on separate molecules, in trans. The bipartite recognition site is indicated by black segments, with an arrowhead to mark its 5 0 -3 0 orientation: strand polarities are also indicated at DNA termini. Cleavage loci 10 and 12 nt distant from the site are marked with vertical arrows: upstream and downstream loci are noted with À or + signs, respectively. (b) The two sites in (a) are both shown bound to one A 2 B protomer of BcgI: an interaction between the protomers is suggested by the double-headed arrow (in red). The B subunit (yellow oval) is responsible for DNA specificity and is positioned spanning both segments of the recognition sequence. The A subunits carry both REase and MTase activities and are illustrated as separate domains connected by a flexible linker. The MTase domains (green squares) overlay the sites of methylation in the specified segments of the recognition sequence. The REase domains (cyan triangles) are placed against the target bonds at the À10 and +10 positions, upstream and downstream of the site in top and bottom strands, respectively: to mark their orientation on the DNA, the triangle points towards the 5 0 -end of each strand. At this stage, BcgI has yet to engage the scissile bonds at the À12 and +12 positions.(c) As (b) except that two additional A 2 B units from free solution now bridge the A 2 B units bound to each site via interactions between the nuclease domains of the free and the DNA-bound units. The two nuclease domains from one of the extra A 2 B units engage the scissile bonds at the À12 positions upstream of both recognition sites (on the left, as drawn in [c]) while those from the other occupy the downstream +12 positions in both sites (on the right). The scheme thus results in a dimer of nuclease domains, with two catalytic centres for phosphodiester hydrolysis at all four loci where BcgI makes a DSB. [/fig]
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Case Report: Creeping Growth in Lymphoplasmacyte-Rich Meningioma—A Radiologic Variant
Lymphoplasmacyte-rich meningioma (LRM) is a rare histologic subtype of meningioma. Creeping-growth pattern is uncommon in meningioma, and the mechanism is unclear. Here, we report a 44-year-old man presented with extremities weakness for 2 months and incontinence for 2 weeks. Head and neck MRI revealed diffuse creeping-growth nodular meningeal masses with skull base, tentorium, sella area, and C1-6 vertebral plane involvement. An operation was carried out, cervical and lower clivus part of the lesion was resected, but gross total resection could not be achieved due to the widespread lesions. Pathologic examination revealed the diagnosis of LRM. The patient is free from progression clinically 3 months postoperatively. We also conducted a systematic literature review about LRM with creeping-growth pattern. A total of only nine cases (including the present case) of creeping-growth LRMs were included and analyzed in terms of clinical manifestations, radiological features, treatment, and outcome. LRMs show a higher rate (7.5%) of creeping-growth pattern than other types of meningiomas. The average creeping length of all creeping-growth LRMs was 11.4 ± 10.9 cm (range, 3-30 cm). Most cases (66.7%) had obvious peritumoral edema. Total removal rate is low (33.3%), and two of them (22.2%) received biopsy, followed by steroids treatment (or further immunosuppressive drugs therapy) and radiotherapy. The recurrence rate is higher than conventional LRMs (22.2 vs. 11.3%), and one patient (11.1%) died 11 months after treatment. Creeping-growth pattern in LRM may be considered as a general radiologic variant. The recurrence rate is higher compared with LRM with round/swelling pattern. We speculated that the pathogenesis of creeping growth in LRM may be associated with damage of lymphatic systems of the central nervous system.
# Introduction
Intracranial meningioma represents the most common primary brain tumor. Radiologically, they frequently behave as swelling dura-based masses. Meningiomas with creepinggrowth pattern are a rare occurrence and are frequently misdiagnosed preoperatively. Lymphoplasmacyte-rich meningioma (LRM) is probably the rarest histologic subtype of meningioma, accounting for around 1% of all intracranial meningiomas. It is characterized by infiltration of lymphoplasmacyte that overshadows the meningioma components. We reported a patient with LRM who presented with diffuse creeping-growth nodular meningeal masses with skull base, tentorium, sella area, and C1-6 vertebral plane involvement radiologically. Eight additional cases of creeping-growth LRM were identified in the literature and were also presented. The mechanism of creeping growth in LRM is unclear. Therefore, we also made a hypothesis to elucidate the mechanism of this rare growth pattern in LRM based on lymphatic systems of the central nervous system (CNS).
## Case presentation
A 44-year-old man presented with extremities weakness for 2 months and incontinence for 2 weeks. He had a history of hospitalization in another hospital 1 month ago. The electromyography testing results of limbs were normal and brain plain MRI showed no obvious abnormalities. A diagnosis of symptomatic parkinsonism (encephalitis-induced most likely) was made, and Madopar was prescribed for 2 weeks, but the symptoms did not alleviate and incontinence occurred. Thus, an indwelling urinary catheter was placed. The dosage of Madopar was gradually increased, but the symptoms did not alleviate. Two weeks later, he was transferred to our hospital. His temperature was 37.6 - C on admission. Physical Abbreviations: CNS, central nervous system; LRM, lymphoplasmacyte-rich meningioma. examination disclosed decreased muscle strength of extremities and Babinski sign positive for lower limbs. White blood count was normal; HIV testing was negative. Head and neck MRI showed creeping-growth isointense lesions on T1-and T2weighted images, compressing the spinal cord. After administration of gadolinium, diffuse creeping-growth nodular meningeal masses with skull base, tentorium, sella area, and C1-6 vertebral plane involvement were detected, compressing medulla and adjacent spinal cord. Routine urine examination of the patient showed that the white blood cell count was 44/HP, and pus cells were detected. The patient received levofloxacin instillation for 7 days and the temperature went normal. A preliminary diagnosis of meningeal inflammatory lesion was made based on radiological manifestations. Subsequently, he underwent an operation. The patient was placed with a left lateral decubitus position, and we used a posterior midline approach. We formed the bone flap (within 4 cm above foramen magnum) and removed posterior arch of C1, performed laminotomy of C2-C6. During operation, creeping-growth subdural masses surrounding the medulla and the spinal cord were detected, with a clear boundary. We mainly used ultrasonic aspiration to achieve tumor debulking and micro-scissors to achieve separation. The cervical and lower clivus part of the lesion was resected; however, gross total resection could not be achieved due to the widespread lesions. Finally, long-segment laminoplasty of C2-C6 and cranioplasty of occipital bone were performed. Postoperative MRI confirmed the total resection of the cervical and lower clivus part of the lesion. The muscle strength of extremities of the patient recovered to normal level, and the Babinski sign was negative for lower limbs 4 days after surgery. Then he discharged and was transferred to our two-way referral hospital. The patient is free from progression clinically 3 months postoperatively. A histological test revealed that the tumor stroma was surrounded by prominent lymphocytes and plasmocytes. The tumor cells were positive for EMA, P63, PR, and negative for GFAP, Oligo2, and CK; lymphocytes were positive for LCA, CD3, and CD20; plasmocytes were positive for CD138, partly positive for IgG and IgG4, which is consistent with an LRM. A multidisciplinary team (Neurosurgery, Neurology, Oncology, and Pathology team) discussion for the patient was performed, and we recommended adjuvant radiotherapy while the patient refused. Thus, we recommended regular imaging follow-up, and if the tumor had sign of progression, stereotactic radiotherapy should be performed and the patient agreed.
## Literature review
This study was conducted and reported according to the Preferred Reporting Items for Systematic Reviews and Meta-Analysis statement.
## Search strategy and article selection
The PubMed and Embase databases were searched for case reports and series relevant to LRM published up to December 31, 2020. "Lymphoplasmacyte, " "rich, " "lymphoplasmacyte-rich, " and "meningioma" were used as either keywords or medical subject headings. In addition, the references cited in selected articles were hand-searched and reviewed to identify additional potentially eligible studies. Titles and abstracts of all articles were screened. Only articles describing cases of creeping-growth LRM were selected. Articles without any radiological imaging were excluded; articles published in languages other than English were also excluded from the study. References from relevant reviews were hand-searched. Subsequently, relevant articles were retrieved and evaluated independently by two authors (JL and XZ). A cross-reference check of the citations of each included relevant article was done to ensure that no relevant studies were missed by the computerized database search. Disagreements regarding the inclusion of studies were resolved by the judgment of a third author (SZ).
## Data extraction
Two authors (JL and XZ) independently extracted the following characteristics from the included studies: setting, study type, demographic data of presented patients, duration, tumor location, radiologic findings, treatment, outcome, and follow-up. The length of creeping growth in cases with complex skull base involvement is determined by the maximal creeping length on the axial, coronal, and sagittal planes.
# Result
Our search identified 46 potential citations for full-text evaluation. Eight cases of creeping-growth LRM from five different publications between 1997 and 2018 could be extracted from the literature and matched the inclusion criteria of our study.
Till now, around 120 cases of LRM have been reported in the English literature, and nine cases (7.5%) of creeping-growth LRM (including the present case) have been reported. The key characteristics of all reported creeping-growth LRM are summarized in . There were five male and four female patients. The mean age was 40.8 ± 12.8 years (range, 22-55 years). Unlike other histologic subtypes of meningiomas, no female predominance was observed (0.8:1), which was similar to the literature report. Most cases (55.6%) were non-skull base masses (Supplementary . The average creeping length of all creeping-growth LRMs was 11.4 ± 10.9 cm (range, 3-30 cm). Most cases (62.5%) had obvious peritumoral edema, higher than that (40%) of round LRMs reported by Tao et al.. Total removal rate is low (33.3%) due to the wide-spread lesion. Two of them (22.2%) received biopsy, followed by steroids treatment (or further immunosuppressive drugs therapy) and radiotherapy. The recurrence rate is higher than conventional LRMs (22.2 vs. 11.3%) and one patient (11.1%) died 11 months after treatment.
# Discussion
Lymphoplasmacyte-rich meningioma was firstly described by Banerjee and Blackwood in 1971, which was characterized by infiltration of lymphoplasmacyte that overshadows the meningioma components. It is probably the rarest histologic subtype of meningioma, accounting for around 1% of all intracranial meningiomas and was classified as WHO grade I tumor. The pathogenesis, radiologic features, and outcome of this subtype of meningioma remain unclear because of their rarity. Here, we report an extremely rare case of LRM with a creeping-growth pattern mimicking idiopathic hypertrophic pachymeningitis and examine previously reported cases of creeping-growth LRM in an attempt to provide an up-to-date summary of the condition and illustrate the possible mechanism of this rare kind of growth pattern.
## Differential diagnosis
For subdural masses with creeping growth, the most common preoperative diagnosis can be a subdural hematoma, lymphoma, dural metastasis, and sarcoma. However, our study suggests that meningiomas should also be included in the differential diagnosis. Subdural LRMs usually have "dural tail sign" and are more likely to show extensive uneven thickening of nearby meninges and peritumoral edema (4). Patients with subdural hematoma usually have a brain-trauma history, with surrounding edema on T2-weighted image and no enhancement on enhanced MRI. Subdural lymphomas tend to be primary ones such as mucosal-associated lymphoid tissue lymphomas, usually with nodular/lobulate borders and leptomeningeal extension. Dural metastasis lesions usually demonstrate hyperintensity on T2-weighted imaging and have a history of systemic primary neoplasm, especially from melanoma, breast, and prostate cancers . Sarcomas tend to affect pediatric or younger patients and are usually accompanied by systemic leukemia. Other differential diagnoses include empyema or granuloma, neurosarcoidosis, tuberculosis, inflammatory pseudotumor, idiopathic hypertrophic pachymeningitis and Rosai-Dorfman disease . Thus, when dealing with subdural masses, neurosurgeons should at least be reminded of the possibility of LRM, and further examinations such as enhanced MRI should be considered which would be helpful for the differential diagnosis.
## Therapeutic strategy
As for treatment of creeping-growth LRM, surgical resection is the optimal treatment modality to achieve pathological diagnosis, alleviate symptoms, and sometimes cure the disease (4, 5). However, the total removal rate is very challenging in some cases because of the wide-creeping lesion, which is hard to expose and resect. As summarized in , two patients had recurrence, and they both received incomplete resection. Thus, we hold that for patients receiving incomplete resection, radiotherapy should be recommended to achieve better tumor control. Hirunwiwatkul et al.performed a biopsy to achieve pathological diagnosis of an LRM extending from planum sphenoidale down along the clivus to the foramen magnum and then prescribed prednisone and later azathioprine, followed by fractionated radiotherapy. And the patient was free from recurrence for over 6 months. Therefore, we hold that for creeping-growth LRM, a comprehensive treatment program (surgery, steroids, and immunosuppressive treatment combined with radiotherapy) could be recommended, especially for those receiving incomplete resection.. Meningeal lymphatic systems are mainly located within the dura mater and carry fluid, macromolecules, and immune cells, such as plasmocytes and lymphocytes, to the draining deep cervical lymph nodes.
## The lymphatic system of cns
## Possible mechanism of creeping-growth pattern in lrm
Radiologically, meningioma frequently manifests as swelling dura-based mass. The creeping-growth pattern of meningiomas is rare; however, LRMs show a higher rate (7.5%) of creeping-growth pattern than other types of meningioma. The mechanism of creeping growth in LRM is unclear. Yamaki et al.hold that microscopically tumor component of LRM is almost replaced by abundant inflammatory cells; thus, LRM may behave differently from conventional meningioma radiologically. Liu et al.suggest that lymphoplasmacytes could form cuff-like structure around the vascular tissue and damage the blood-brain barrier, thus causing enhancement on post-contrast MRI. We initially elucidate this rare phenomenon based on the lymphatic systems of the CNS.
We speculated that the mechanism of creeping growth in LRM might be as follows: (1) meningioma grows and erodes the meningeal lymphatic systems; (2) meningioma may even keep growing and transmitting to adjacent dura mater through the highway of the lymphatic system. (3) meningiomas express chemotactic agents such as inflammatory and chemotactic cytokines to assemble lymphocytes and plasmocytes; (4) vast lymphocytes and plasmocytes subsequently keep leaking from the lymphatic structure, overshadowing the meningioma components; and (5) lymphocytes and plasmocytes change the microenvironment of the tumor and prevent the gathering and adhering of meningioma components or destroy/hamper the formation of peritumoral fibrin, restraining the swelling growth of the tumor, and thus LRM tends to grow diffusely along the meninges. In our case, the meningioma grows as continuous multiple nodular pattern probably under the influence of prominent infiltrated lymphoplasmacytes instead of forming a regular shape (swelling/round) of a meningioma. In addition, the margin of case 4 (Supplementary . These cases indicated that the pathogenesis of LRM might be associated with the lymphatic system, especially the wider lymphatic vessels alongside intracranial venous sinuses. For the case treated in our hospital reported by , the whole intracranial dura mater was involved, which indicated the meningioma components may be transmitted through the meningeal lymphatic system to reach such widespread meninges. As for those LRMs behaving as swelling pattern, the destruction of the meningeal lymphatic systems may occur while the tumor is relatively large, whereas the infiltrated immune cells could not reshape the tumor.
The hypothesis that the pathogenesis of LRM associates with CNS lymphatic system damage could interpret the following four aspects of LRM behaviors. (1) Massive lymphocytes and plasmocytes infiltrating the tumor is uncommon, which means they are most likely to originate from lymphatic systems rather than blood vessels or cerebrospinal fluid. (2) Many creeping-growth LRMs could reach a creeping length of over 10 cm. The extraordinary diffuse involvement of dura mater may be attributed to the lymphatic system pathway transfusion. (3) It could explain why LRM may behave as regular (swelling/round) shape or a relatively higher rate (around 10%) of creeping-growth pattern than other histologic subtypes of meningiomas due to the different stages of lymphatic system damage. (4) The lymph proteins leaking from lymphatic systems or the dysfunction of cerebrospinal fluid absorbing caused by lymphatic destruction could further induce brain edema, which is compatible with the high rate (62.5%) of severe peritumoral edema in creeping-growth LRM.
The strength of our study is that we reviewed all the cases of creeping-growth LRMs and concluded that LRMs show a higher rate of creeping growth compared with other histological meningiomas; thus, the creeping-growth pattern in LRMs may be considered as a common radiologic variant. In addition, we proposed a hypothesis that the pathogenesis of LRMs may be associated with damage of lymphatic systems, which could nicely explain why LRM may behave as regular (swelling/round) shape or a relatively higher rate (around 10%) of creeping-growth pattern than other histologic subtypes of meningiomas due to the different stage of lymphatic system damage. The limitations of our study are retrospective in nature. Furthermore, because of financial limitations and local medical insurance policy restrictions, we could not obtain sufficient data regarding genetic conditions. Moreover, some cases of the literature were lost to follow-up. Finally, no evidence from the experiments confirming the mechanism of creeping growth of LRM existed. Therefore, further investigations, such as tracing the origin of lymphoplasmacytes, analyzing the function of infiltrated lymphocytes and plasmocytes, the possible chemotactic agents expressed by LRM, and exploring genetic differences, are necessary to elucidate the preferred mechanisms of creeping growth in LRM.
# Conclusion
Lymphoplasmacyte-rich meningiomas show a higher rate (7.5%) of creeping-growth pattern than other types of meningiomas and may be considered as a general radiologic variant. Currently, though total removal is challenging, surgical resection is the optimal treatment to cure the disease and relieve the symptoms. The recurrence rate is higher compared with LRM with round/swelling pattern. We proposed a hypothesis that the pathogenesis of creeping growth in LRM may be associated with damage of the lymphatic systems of the CNS.
# Data availability statement
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.
# Ethics statement
The studies involving human participants were reviewed and approved by Ethics Committee of West China Hospital of Sichuan University. Written informed consent was obtained from the patient and his legal representatives for the publication. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.
# Author contributions
XZ, SZ, and WL contributed to the conception and design of the study. JL, MF, and XD developed the methodology. JL, XZ, MF, XD, SZ, and WL helped in the acquisition and analysis of data. JL, XZ, and MF involved in writing, reviewing, and revision of the manuscript. MF and WL provided the technical and material support. SZ and WL supervised the study. All authors have read and approved the manuscript. |
Selective Devaluation Affects the Processing of Preferred Rewards
Supplementary Analysis 1: Link between BMI and selective devaluation effects Supplementary Figure S12: Effect of MPO over LPO in the devaluation condition 17 Supplementary Figure S13: Effect of MPO over HPO in the devaluation condition 18 References 19
In light of studies byand [bib_ref] Loss of lateral prefrontal cortex control in food-directed attention and goaldirected food..., Janssen [/bib_ref] who found a decreased behavioural selective devaluation effect with higher BMI, we conducted an exploratory correlational analysis between the BMI and the selective devaluation effect. For the selective devaluation effect, we used the mean amplitude of the devaluationnondevaluation difference signal for the HPO outcome in the time windows yielded by the cluster-based analysis for the difference signal (300-470 and 480-600 ms) for electrodes Fz, FCz, Cz, and Pz. This analysis yielded two significant correlations, one at electrode site Cz for the early cluster time window (r = .37, p = .042) and the second at FCz for the late cluster time (r = .36, p = .045). Complete inferential statistics and descriptive data can be found in below. These correlations are consistent with a decreased selective devaluation effect in the P300 with increasing BMI. As we retrieved the BMI from participants' statements of height and weight, which are known to be imprecise [bib_ref] Validity of self-reported height and weight in 4808 EPIC-Oxford participants, Spencer [/bib_ref] , and since the present sample included only few overweight (n = 7) and obese participants (n = 2), future studies will need to explore whether this effect prevails with more objective BMI measures and a better fitting sample. . BMI according to subjects' estimation of their weight and height plotted against difference in mean amplitude (devaluation -nondevaluation) in the cluster time windows (300-470 and 480-600 ms) for Fz, FCz, Cz, and Pz. Regression line is plotted in red with a grey ribbon for the confidence interval.
To see whether we could replicate the findings of Peterburs, Sannemann, and
Bellebaum (2019), we took into consideration all clusters for the analysis of the outcome type effect in the nondevaluation condition which are reported below.
Cluster-based permutation tests for the outcome type main effect in the nondevaluation condition revealed significant differences within outcome types (p < .001; for topographical plots, see [fig_ref] Figure S4: showing more positive amplitudes for the medium preference outcome [/fig_ref]. This effect was evident in two clusters, of which the additionally included centroparietal electrodes inconsistently (see [fig_ref] Figure S6: Clusters [/fig_ref].
Amplitudes were also significantly more positive for the HPO compared to the MPO (p = .003). This effect was evident in clusters covering only later time windows. One cluster covered a time window between 300 and 420 ms at frontal, frontocentral, central, centroparietal, and parietal electrodes, while a second cluster was found from 480 and 590 ms at frontocentral, central, centroparietal, and parietal sites (see [fig_ref] Figure S7: Clusters [/fig_ref].
[fig] 3 SupplementaryFigure S1 4 Supplementary Analysis 2 5 SupplementaryFigure S2 9 SupplementaryFigure S5 12 SupplementaryFigure S8 13 SupplementaryFigure S9 14 SupplementaryFigure S10: Correlation between BMI and cluster amplitude Replication of outcome type effect reported in Peterburs et al. (2019) Outcome type main effect 7 Supplementary Figure S3: Effect of HPO over LPO for average amplitudes 8 Supplementary Figure S4: Effect of MPO over LPO for average amplitudes Interaction effect of outcome type and valuation condition 10 Supplementary Figure S6: Outcome type effect in the nondevaluation condition 11 Supplementary Figure S7: Effect of HPO over LPO in the nondevaluation condition Effect of MPO over LPO in the nondevaluation condition Effect of HPO over MPO in the nondevaluation condition Outcome type effect in the devaluation condition 15 Supplementary Figure S11: Effect of HPO over LPO in the devaluation condition 16 Supplementary Analysis 1 [/fig]
[fig] Figure S2: Clusters (90-200 and 240-600 ms) showing significant modulation of outcome processing by outcome type. Topographical plots represent time series of F-values at each time point and channel. Stars indicate electrodes included in the cluster. [/fig]
[fig] Figure S3: showing more positive amplitudes for the high preference outcome (HPO) over the low preference outcome (LPO) for the mean amplitude between the devaluation and nondevaluation condition. Topographical plots represent time series of t-values at each time point and channel. Stars indicate electrodes included in the cluster. [/fig]
[fig] Figure S4: showing more positive amplitudes for the medium preference outcome (MPO) over the low preference outcome (LPO) for the mean amplitude between the devaluation and nondevaluation condition. Topographical plots represent time series of t-values at each time point and channel. Stars indicate electrodes included in the cluster. [/fig]
[fig] Figure S5: Clusters (300-470 and 480-600 ms) showing significant modulation of outcome processing by outcome type in the devaluation-nondevaluation difference signal. Topographical plots represent time series of F-values at each time point and channel. Stars indicate electrodes included in the cluster. [/fig]
[fig] Figure S6: Clusters (150-200 and 250-600 ms) showing an effect of outcome type in the nondevaluation condition. Topographical plots represent time series of F-values at each time point and channel. Stars indicate electrodes included in the cluster. [/fig]
[fig] Figure S7: Clusters (150-200 and 250-600 ms) showing more positive amplitudes for the high preference outcome (HPO) over the low preference outcome (LPO) for the nondevaluation condition. Topographical plots represent time series of t-values at each time point and channel. Stars indicate electrodes included in the cluster. [/fig]
[fig] Figure S8: showing more positive amplitudes for the medium preference outcome (MPO) over the low preference outcome (LPO) for the nondevaluation condition. Topographical plots represent time series of t-values at each time point and channel. Stars indicate electrodes included in the cluster. [/fig]
[fig] Figure S9: showing more positive amplitudes for the high preference outcome (HPO) over the medium preference outcome (MPO) for the nondevaluation condition. Topographical plots represent time series of t-values at each time point and channel. Stars indicate electrodes included in the cluster. [/fig]
[fig] Figure S10: showing an effect of outcome type in the devaluation condition. Topographical plots represent time series of F-values at each time point and channel. Stars indicate electrodes included in the cluster. [/fig]
[fig] Figure S11: Clusters (300-470 and 480-540 ms) showing more positive amplitudes for the high preference outcome (HPO) over the low preference outcome (LPO) for the devaluation condition. Topographical plots represent time series of t-values at each time point and channel. Stars indicate electrodes included in the cluster. [/fig]
[fig] Figure S12: Clusters (300-470 and 480-590 ms) showing more positive amplitudes for the medium preference outcome (MPO) over the low preference outcome (LPO) for the devaluation condition. Topographical plots represent time series of t-values at each time point and channel. Stars indicate electrodes included in the cluster. [/fig]
[fig] Figure S13: Cluster (340-400 ms) showing more positive amplitudes for the medium preference outcome (MPO) over the high preference outcome (HPO) for the devaluation condition. Topographical plots represent time series of t-values at each time point and channel. Stars indicate electrodes included in the cluster. [/fig]
|
Synthetic Strategies for 5- and 6-Membered Ring Azaheterocycles Facilitated by Iminyl Radicals
The totality of chemical space is so immense that only a small fraction can ever be explored. Computational searching has indicated that bioactivity is associated with a comparatively small number of ring-containing structures. Pyrrole, indole, pyridine, quinoline, quinazoline and related 6-membered ring-containing aza-arenes figure prominently. This review focuses on the search for fast, efficient and environmentally friendly preparative methods for these rings with specific emphasis on iminyl radical-mediated procedures. Oxime derivatives, particularly oxime esters and oxime ethers, are attractive precursors for these radicals. Their use is described in conventional thermolytic, microwave-assisted and UV-vis based preparative procedures. Photoredox-catalyzed protocols involving designer oxime ethers are also covered. Choice can be made amongst these synthetic strategies for a wide variety of 5-and 6-membered ring heterocycles including phenanthridine and related aza-arenes. Applications to selected natural products and bioactive molecules, including trispheridine, vasconine, luotonin A and rutaecarpine, are included. Molecules 2016, 21, 660 2 of 25Thus, the development of synthetic strategies for the ring systems associated with bioactivity that are fast, efficient and of low environmental impact, deserves special attention.The advantages of free-radical based preparative methods include the usually neutral conditions, the tolerance for many unprotected functional groups and the availability of much kinetic, thermodynamic and mechanistic data to guide the design of experimental methodology. During the last two decades a great deal of research has been directed towards making radical-mediated synthetic methods safer, more efficient and more convenient[8][9][10][11][12][13]. New tactics have been devised for avoiding hazardous initiator peroxides or azo-compounds and for dispensing with toxic tin, mercury, copper and other metal reagents. For example, 'pro-aromatic' reagents, based on the cyclohexadiene structure, release many radical types without the need for metals[14]. Murphy and co-workers' development of organic super electron donors unlocked completely new ways of generating radicals and radical-ions and harnessing them synthetically[15][16][17]. The unique properties of organoboron compounds have led to the design of several different reagent types for radical release including B-alkylcatecholboranes[18,19]and N-heterocyclic carbene boranes[20][21][22][23]. The discovery of homogeneous photoredox catalysts (PCs) has had huge impact on radical-mediated preparations. The most popular are complexes of Ru or Ir [24-27] that re-introduce metals, albeit in small quantities. However, organic dyes and other donor molecules are also coming into use as PCs[28,29]. Heterogeneous photoredox catalysts, particularly titanium dioxide (titania, TiO 2 ), possess the added convenience of easy removal after use by filtration or centrifugation. Their exploitation for radical mediated preparations is also developing rapidly[30][31][32].The N-O bonds in oximes and in oxime derivatives are comparatively weak and break homolytically with production of a pair of N-and O-centered radicals. Aldehydes and ketones are available as starting materials in huge variety from natural and commercial sources. Oximes can be prepared essentially quantitatively from them simply by treatment with hydroxylamine hydrochloride. The consequence is that oxime esters of many types containing the >C=N-OC(O)-structural unit (carbonyl oximes) and oxime ethers containing the >C=N-O-unit are very readily accessible. Most members of both classes are stable to moderate heat and hydrolysis, are non-toxic and non-hazardous, are easily handled and have long shelf lives. Suitably functionalised, they have proved amazingly adaptable for radical generation by an unprecedentedly wide range of methods[33]. These include conventional thermolyses, microwave irradiations, UV photolyses, sensitised UV photolyses and with several types of photoredox catalysis. Oxime derivatives are therefore particularly flexible, convenient and benign and stand as very attractive alternatives to other more hazardous radical precursors. This article reviews the use of both carbonyl oximes and oxime ethers in radical mediated organic syntheses.When fittingly stimulated, both compound types initially yield N-centred iminyl radicals >C=N ‚ (Im, Scheme 1). Those suitably accoutred with acceptor groups can, when appropriately manipulated, yield azaheterocycles. An O-centred radical [ ‚ OC(O)Z] is released from a carbonyl oxime together with the iminyl radical and can be chosen to end up as volatile or otherwise easily separable by-products. For the oxime ether precursors, best results are usually achieved with O-aryl substituents. In this case the by-product is usually a phenol (ArOH) which can readily be removed because of its mild acidity. Iminyl radicals with butene or butyne type side chains selectively undergo 5-exo cyclisation to produce 5-member ring containing dihydropyrrole type products. By way of contrast, iminyl radicals with aromatic or heteroaromatic acceptor substituents preferentially yield 6-membered ring pyridine, quinoline etc. products. In some instances this results from an initial 5-exo spiro cyclization followed by ring expansion via an aziridinyl type intermediate (see for example Section 4.1). Preparations of many different azaheterocycle types may therefore be achieved by careful choice of the acceptor substituent(s), and by tuning the reaction conditions and methodology. Molecules 2016, 21, 660 3 of 25 Molecules 2016, 21, 660 3 of 23 Scheme 1. Generation of iminyl radicals from oxime derivatives and subsequent ring closures.Syntheses of Dihydropyrroles and PyrrolesOrganotin promoted radical methodology is justly famed because it works seamlessly in so many situations and has proved so dependable. Many ingenious syntheses of azaheterocycles have employed organotin hydrides or ditins for the generation of iminyl or aminyl radicals. Zard, for example, described tin hydride-mediated syntheses of dihydropyrroles, indolizidines and other aza-heterocycles from sulfenimines (PhS-N=C<), thionocarbazones and other derivatives[34,35]. Nanni and co-workers generated iminyl radicals by ring closures of C-centred radicals onto organic nitriles and hence prepared many heterocyclic systems. They also employed tin-free thermolytic and other processes[36][37][38]. Much of this earlier research has been reviewed by Bowman and Aldabbagh[39][40][41]and/or by Fallis and Brinza[42]. Recently, Zhang and Studer have published an outstanding review of aza-arene syntheses flowing from radical additions to organic isonitriles[43]. This methodology exploits the formation and ring closures of amidoyl radicals (Ar-N=C - -R).Pyrrole and Dihydropyrrole Preparations from Carbonyl OximesNumerous alkaloids contain pyrrole, dihydropyrrole or related rings and many biological roles are associated with these structures[44][45][46]. Oxime esters 1 can easily be prepared from oximes reacting with either carboxylic acids or acyl halides[47,48]. On photolysis they release an iminyl radical together with an acyloxyl radical and the latter rapidly extrudes CO2 with production of a C-centred radical[48,49](Scheme 2). Rodrigues, Sampedro and co-workers used acyl oximes such as 2 as efficient sources of iminyl radicals[50][51][52]. With this precursor type, the radical co-produced with the iminyl during UV photolyses was acyloxyl [CH3CO2 - ] that simply furnished volatile CH4 and CO2 as by-products. They reported that the iminyls could be conscripted into syntheses of many types of heterocycles including dihydropyrroles. UV photolysis of 2 through Pyrex released iminyl radical 3 with an alkenyl side chain. These selectively cyclised in the 5-exo mode with production of pyrolidinylmethyl radical 4 that subsequently abstracted an H-atom from co-reactant cyclohexa-1,4diene (CHD) to yield 3,4-dihydropyrrole derivative 5 (Scheme 2). Scheme 2. Photo-dissociation of oxime esters and preparation of dihydropyrroles[48,51]. Molecules 2016, 21, 660 4 of 25the iminyl during UV photolyses was acyloxyl [CH3CO2 - ] that simply furnished volatile CH4 and CO2 as by-products. They reported that the iminyls could be conscripted into syntheses of many types of heterocycles including dihydropyrroles. UV photolysis of 2 through Pyrex released iminyl radical 3 with an alkenyl side chain. These selectively cyclised in the 5-exo mode with production of pyrolidinylmethyl radical 4 that subsequently abstracted an H-atom from co-reactant cyclohexa-1,4diene (CHD) to yield 3,4-dihydropyrrole derivative 5 (Scheme 2). Scheme 2. Photo-dissociation of oxime esters and preparation of dihydropyrroles[48,51].Dioxime oxalates 8 are another type of oxime ester that proved convenient for clean generation of iminyl radicals. Symmetrical types were made by treatment of an oxime 6 with oxalyl chloride to yield oxime oxalyl chlorides 7 as intermediates (Scheme 3). Although these could be isolated, they hydrolysed and degraded quickly, so immediate treatment with another equivalent of either the same Scheme 2. Photo-dissociation of oxime esters and preparation of dihydropyrroles[48,51].
# Introduction
Several recent articles have drawn attention to the virtually boundless extent of chemical space; the domain that contains the totality of all possible compounds [bib_ref] The chemical space project, Reymond [/bib_ref] [bib_ref] Bioactivity-guided navigation of chemical space, Bon [/bib_ref]. The total number of molecules that could be made from only 30 atoms is in the range 10 20 to 10 [bib_ref] Visible light photocatalysis as a greener approach to photochemical synthesis, Yoon [/bib_ref] [bib_ref] Cheminformatics analysis of organic substituents: Identification of the most common substituents, calculation..., Ertl [/bib_ref] , "drug-like" chemical space comprises over 10 60 molecules [bib_ref] Experimental and computational approaches to estimate solubility and permeability in drug discovery..., Lipinski [/bib_ref] [bib_ref] The art and practice of structure-based drug design: A molecular modelling perspective, Bohacek [/bib_ref] and, of course, even these huge numbers are insignificant in comparison with the protein or nucleic acid spaces. The number of polypeptide chains of modest (250 unit) length, drawn from the 20 natural amino acids, exceeds the 'trans-astronomical' number of 10 325 [bib_ref] Self-organization of matter and the evolution of biological macromolecules, Eigen [/bib_ref]. The CAS registry currently contains about 10 8 chemical substances. Its present rate of growth is about 5ˆ10 [bib_ref] Self-organization of matter and the evolution of biological macromolecules, Eigen [/bib_ref] substances per year, so that at this rate more than 10 54 years would be needed just to explore "drug-like" chemical space! It is abundantly evident that only a minute fraction of chemical space can ever be preparatively accessed. To address this problem, computational algorithms are being devised capable of virtual screening and/or for locating, within the total space, "islands" or "trees" of substances with potentially desirable properties such as bioactivity [bib_ref] The chemical space project, Reymond [/bib_ref] [bib_ref] Bioactivity-guided navigation of chemical space, Bon [/bib_ref] [bib_ref] Quest for the rings. In silico exploration of ring universe to identify..., Ertl [/bib_ref]. Ertl and co-workers developed self-organising neural networks which showed that a comparatively small number of ring structural units is associated with bioactivity. They listed 30 heterocyclic moieties as of crucial importance and 22 of these contained one or more N-atoms [bib_ref] Quest for the rings. In silico exploration of ring universe to identify..., Ertl [/bib_ref]. Pyrrole, indole and related structures figured prominently, as did pyridine, quinoline, quinazoline and analogous 6-membered ring-containing aza-arenes. The immense size of chemical space presents exciting opportunities of discovering hitherto unknown and extraordinary substances with properties beneficial to human society. Its size also represents a huge challenge for preparative chemists such that it is imperative to open up every possible avenue that might facilitate the task. The exploration and exploitation of identified "islands" depends critically, of course, on the availability of practical preparative methods.
Dioxime oxalates 8 are another type of oxime ester that proved convenient for clean generation of iminyl radicals. Symmetrical types were made by treatment of an oxime 6 with oxalyl chloride to yield oxime oxalyl chlorides 7 as intermediates (Scheme 3). Although these could be isolated, they hydrolysed and degraded quickly, so immediate treatment with another equivalent of either the same This review also focuses on the iminyl radical based synthetic methodology developed for the sets of aza-arenes identified above as of importance in the bioactivity islands of chemical space.
## Syntheses of dihydropyrroles and pyrroles
Organotin promoted radical methodology is justly famed because it works seamlessly in so many situations and has proved so dependable. Many ingenious syntheses of azaheterocycles have employed organotin hydrides or ditins for the generation of iminyl or aminyl radicals. Zard, for example, described tin hydride-mediated syntheses of dihydropyrroles, indolizidines and other aza-heterocycles from sulfenimines (PhS-N=C<), thionocarbazones and other derivatives [bib_ref] Recent progress in the generation and use of nitrogen-centred radicals, Zard [/bib_ref] [bib_ref] Generation and capture of iminyl radicals from ketoxime xanthates, Gagosz [/bib_ref]. Nanni and co-workers generated iminyl radicals by ring closures of C-centred radicals onto organic nitriles and hence prepared many heterocyclic systems. They also employed tin-free thermolytic and other processes [bib_ref] On the regioselectivity of imidoyl radical cyclisations, Nanni [/bib_ref] [bib_ref] Reactions of 1-(2-alkoxyphenyl)alkaniminyl radicals, Leardini [/bib_ref] [bib_ref] From azides to nitrogen-centered radicals: Applications of azide radical chemistry to organic..., Minozzi [/bib_ref]. Much of this earlier research has been reviewed by Bowman and Aldabbagh [bib_ref] Synthesis of heterocycles by radical cyclization, Aldabbagh [/bib_ref] [bib_ref] Bu 3 SnH mediated oxidative radical cyclization onto imidazoles and pyrroles, Aldabbagh [/bib_ref] [bib_ref] Synthesis of heteroarenes via radical cyclisation onto nitriles, Bowman [/bib_ref] and/or by Fallis and Brinza [bib_ref] Free radical cyclizations involving nitrogen, Fallis [/bib_ref]. Recently, Zhang and Studer have published an outstanding review of aza-arene syntheses flowing from radical additions to organic isonitriles [bib_ref] Recent advances in the synthesis of nitrogen heterocycles via radical cascade reactions..., Zhang [/bib_ref]. This methodology exploits the formation and ring closures of amidoyl radicals (Ar-N=C ‚ -R).
## Pyrrole and dihydropyrrole preparations from carbonyl oximes
Numerous alkaloids contain pyrrole, dihydropyrrole or related rings and many biological roles are associated with these structures [bib_ref] Privileged structures as leads in medicinal chemistry, Costantino [/bib_ref] [bib_ref] Synthesis and biological activity of pyrrole, pyrroline and pyrrolidine derivatives with two..., Bellina [/bib_ref] [bib_ref] Living on pyrrolic foundations -advances in natural and artificial bioactive pyrrole derivatives, Domagala [/bib_ref]. Oxime esters 1 can easily be prepared from oximes reacting with either carboxylic acids or acyl halides [bib_ref] Reactions of diene-conjugated 1,3-dipolar intermediates: A versatile and efficient route to dibenz[c,e]azepines..., Cullen [/bib_ref] [bib_ref] Exploitation of aldoxime esters as radical precursors in preparative and EPR spectroscopic..., Mccarroll [/bib_ref]. On photolysis they release an iminyl radical together with an acyloxyl radical and the latter rapidly extrudes CO 2 with production of a C-centred radical [bib_ref] Exploitation of aldoxime esters as radical precursors in preparative and EPR spectroscopic..., Mccarroll [/bib_ref] [bib_ref] Enhanced radical delivery from aldoxime esters for EPR and ring closure applications, Mccarroll [/bib_ref] (Scheme 2). Rodrigues, Sampedro and co-workers used acyl oximes such as 2 as efficient sources of iminyl radicals [bib_ref] New light-induced iminyl radical cyclization reactions of acyloximes to isoquinolines, Alonso [/bib_ref] [bib_ref] Photocyclization of iminyl radicals: Theoretical study and photochemical aspects, Alonso [/bib_ref] [bib_ref] Photochemistry of acyloximes: Synthesis of heterocycles and natural products, Alonso [/bib_ref]. With this precursor type, the radical co-produced with the iminyl during UV photolyses was acyloxyl [CH [bib_ref] Cheminformatics analysis of organic substituents: Identification of the most common substituents, calculation..., Ertl [/bib_ref] ‚ ] that simply furnished volatile CH [bib_ref] Experimental and computational approaches to estimate solubility and permeability in drug discovery..., Lipinski [/bib_ref] and CO 2 as by-products. They reported that the iminyls could be conscripted into syntheses of many types of heterocycles including dihydropyrroles. UV photolysis of 2 through Pyrex released iminyl radical 3 with an alkenyl side chain. These selectively cyclised in the 5-exo mode with production of pyrolidinylmethyl radical 4 that subsequently abstracted an H-atom from co-reactant cyclohexa-1,4-diene (CHD) to yield 3,4-dihydropyrrole derivative 5 (Scheme 2). Dioxime oxalates 8 are another type of oxime ester that proved convenient for clean generation of iminyl radicals. Symmetrical types were made by treatment of an oxime 6 with oxalyl chloride to yield oxime oxalyl chlorides 7 as intermediates (Scheme 3). Although these could be isolated, they hydrolysed and degraded quickly, so immediate treatment with another equivalent of either the same oxime 6, or a different oxime, yielded the symmetrical dioxime oxalates 8 or unsymmetrical types such as 11a-d respectively [bib_ref] Dioxime oxalates; new iminyl radical precursors for syntheses of N-heterocycles, Portela-Cubillo [/bib_ref]. Dioxime oxalates 8 were "clean" and atom-efficient because, apart from CO 2 , they delivered only iminyl radicals 9 on UV photolysis. Photo-dissociations were aided by inclusion of 4-methoxyacetophenone (MAP) as photosensitizer and by the presence of an aryl substituent adjacent to the C=N bond. oxime 6, or a different oxime, yielded the symmetrical dioxime oxalates 8 or unsymmetrical types such as 11a-d respectively [bib_ref] Dioxime oxalates; new iminyl radical precursors for syntheses of N-heterocycles, Portela-Cubillo [/bib_ref]. Dioxime oxalates 8 were "clean" and atom-efficient because, apart from CO2, they delivered only iminyl radicals 9 on UV photolysis. Photo-dissociations were aided by inclusion of 4-methoxyacetophenone (MAP) as photosensitizer and by the presence of an aryl substituent adjacent to the C=N bond. 3,4-Dihydropyrrole derivatives 10 were obtained in moderate yields with toluene acting as both solvent and H-atom donor (Scheme 3). Unsymmetrical dioxime oxalates such as 11 incorporated an aryl-oxime unit, to promote photo-dissociation, as well as an alkenic acceptor unit. These precursors enabled non-aromatic dihydropyrroles such as 12a,b to be accessed. Attempts to convert the latter to pyrolizidines 13 and indolizidines 14 were unsuccessful.
## Pyrrole and dihydropyrrole preparations from oxime ethers
Thermal preparative methods are often superior because of their simplicity and non-hazardous nature. O-Phenyl oxime ethers 15 can easily be made by treatment of carbonyl compounds with the commercially available O-phenylhydroxylamine hydrochloride. Conventional thermolyses of appropriate derivatives were shown to provide dialkyl-or diaryl-iminyl radicals [bib_ref] Thermolyses of O-phenyl oxime ethers. A new source of iminyl radicals and..., Blake [/bib_ref]. Subsequently it was established that microwave heating (μwave) was a particularly efficient means of releasing iminyl radicals and mediating dihydropyrrole preparations [bib_ref] Microwave-assisted preparations of dihydropyrroles from alkenone O-phenyl oximes, Portela-Cubillo [/bib_ref] [bib_ref] Microwave-assisted syntheses of N-heterocycles using alkenone-, alkynone-and aryl-carbonyl O-phenyl oximes: Formal synthesis..., Portela-Cubillo [/bib_ref].
The optimum procedure utilized toluene as both solvent and H-donor together with an equivalent of the ionic liquid (IL) 1-ethyl-3-methyl-1H-imidazol-3-ium hexafluorophosphate (emimPF6) to promote microwave absorbance. This method enabled ketones with but-3-enyl type side chains to be converted to dihydropyrroles 16 in good yields in two steps (Scheme 4). The phenoxyl radicals released from 15 also abstracted H-atoms from the solvent to afford phenol as an easily separable by-product. When oxime ether 17 with an alkyne side chain was microwave irradiated under similar conditions, pyrrole 19 was isolated in good yield. Evidently the first-formed methylene-dihydropyrrole 18 rearranged under the reaction conditions.
Castle and co-workers prepared a set of alkyne-substituted oxime ethers 20 and carried out Scheme 3. Preparation of dioxime oxalates and their use in dihydropyrrole syntheses [bib_ref] Dioxime oxalates; new iminyl radical precursors for syntheses of N-heterocycles, Portela-Cubillo [/bib_ref].
3,4-Dihydropyrrole derivatives 10 were obtained in moderate yields with toluene acting as both solvent and H-atom donor (Scheme 3). Unsymmetrical dioxime oxalates such as 11 incorporated an aryl-oxime unit, to promote photo-dissociation, as well as an alkenic acceptor unit. These precursors enabled non-aromatic dihydropyrroles such as 12a,b to be accessed. Attempts to convert the latter to pyrolizidines 13 and indolizidines 14 were unsuccessful.
## Pyrrole and dihydropyrrole preparations from oxime ethers
Thermal preparative methods are often superior because of their simplicity and non-hazardous nature. O-Phenyl oxime ethers 15 can easily be made by treatment of carbonyl compounds with the commercially available O-phenylhydroxylamine hydrochloride. Conventional thermolyses of appropriate derivatives were shown to provide dialkyl-or diaryl-iminyl radicals [bib_ref] Thermolyses of O-phenyl oxime ethers. A new source of iminyl radicals and..., Blake [/bib_ref]. Subsequently it was established that microwave heating (µwave) was a particularly efficient means of releasing iminyl radicals and mediating dihydropyrrole preparations [bib_ref] Microwave-assisted preparations of dihydropyrroles from alkenone O-phenyl oximes, Portela-Cubillo [/bib_ref] [bib_ref] Microwave-assisted syntheses of N-heterocycles using alkenone-, alkynone-and aryl-carbonyl O-phenyl oximes: Formal synthesis..., Portela-Cubillo [/bib_ref].
The optimum procedure utilized toluene as both solvent and H-donor together with an equivalent of the ionic liquid (IL) 1-ethyl-3-methyl-1H-imidazol-3-ium hexafluorophosphate (emimPF 6 ) to promote microwave absorbance. This method enabled ketones with but-3-enyl type side chains to be converted to dihydropyrroles 16 in good yields in two steps (Scheme 4). The phenoxyl radicals released from 15 also abstracted H-atoms from the solvent to afford phenol as an easily separable by-product. When oxime ether 17 with an alkyne side chain was microwave irradiated under similar conditions, pyrrole 19 was isolated in good yield. Evidently the first-formed methylene-dihydropyrrole 18 rearranged under the reaction conditions.
## Photoredox catalyzed preparations of pyrroles and dihydropyrroles from oxime derivatives
Photoredox catalytic methodology [bib_ref] Visible light photoredox catalysis: Applications in organic synthesis, Narayanam [/bib_ref] [bib_ref] Visible light photoredox catalysis with transition metal complexes: Applications in organic synthesis, Prier [/bib_ref] [bib_ref] Visible light photoredox-controlled reactions of N-radicals and radical ions, Chen [/bib_ref] has also been developed for production of dihydropyrroles from O-aryl oxime ethers substituted with electron withdrawing groups (EWG) in their O-aryl units. Narasaka and co-workers prepared oxime ethers 23 containing 4-CN (or 2,4-di-NO2 or 4-CF3) aryl substituents. On inclusion of a catalytic amount of 1,5-dimethoxynaphthalene (DMN) and irradiation with UV light in 1,4-cyclohexadiene, dihydropyrroles 25 were isolated in good yields (Scheme 5) [bib_ref] Photochemical transformation of γ,δ-unsaturated ketone O-(p-cyanophenyl)oximes to 3,4-dihydro-2H-pyrrole derivatives, Mikami [/bib_ref]. The incident light raised the photocatalyst to an excited triplet state (PC*) that then transferred an electron to the oxime ethers with production of the radical anions 24 (Scheme 5). Loss of the stable phenolate type anions then occurred with release of the corresponding iminyl radicals that subsequently underwent 5-exo cyclisation and H-atom transfer with CHD to afford dihydropyrroles 25. Scheme 4. Preparation of oxime ethers and microwave-promoted syntheses of dihydropyrroles and pyrroles [bib_ref] Microwave-assisted preparations of dihydropyrroles from alkenone O-phenyl oximes, Portela-Cubillo [/bib_ref] [bib_ref] Microwave-assisted syntheses of N-heterocycles using alkenone-, alkynone-and aryl-carbonyl O-phenyl oximes: Formal synthesis..., Portela-Cubillo [/bib_ref] [bib_ref] Microwave-promoted tin-free iminyl radical cyclization with TEMPO trapping: A practical synthesis of..., Cai [/bib_ref].
Castle and co-workers prepared a set of alkyne-substituted oxime ethers 20 and carried out microwave irradiations of mixtures with tetramethylpiperidine-N-oxide (TEMPO) in benzotrifluoride solvent [bib_ref] Microwave-promoted tin-free iminyl radical cyclization with TEMPO trapping: A practical synthesis of..., Cai [/bib_ref]. The ring closed radicals were trapped by the TEMPO with production of intermediates 21 (Scheme 4). These also rearranged, with loss of a piperidinyl radical, so providing 2-acylpyrroles 22 in good to excellent yields.
## Photoredox catalyzed preparations of pyrroles and dihydropyrroles from oxime derivatives
Photoredox catalytic methodology [bib_ref] Visible light photoredox catalysis: Applications in organic synthesis, Narayanam [/bib_ref] [bib_ref] Visible light photoredox catalysis with transition metal complexes: Applications in organic synthesis, Prier [/bib_ref] [bib_ref] Visible light photoredox-controlled reactions of N-radicals and radical ions, Chen [/bib_ref] has also been developed for production of dihydropyrroles from O-aryl oxime ethers substituted with electron withdrawing groups (EWG) in their O-aryl units. Narasaka and co-workers prepared oxime ethers 23 containing 4-CN (or 2,4-di-NO 2 or 4-CF 3 ) aryl substituents. On inclusion of a catalytic amount of 1,5-dimethoxynaphthalene (DMN) and irradiation with UV light in 1,4-cyclohexadiene, dihydropyrroles 25 were isolated in good yields (Scheme 5) [bib_ref] Photochemical transformation of γ,δ-unsaturated ketone O-(p-cyanophenyl)oximes to 3,4-dihydro-2H-pyrrole derivatives, Mikami [/bib_ref]. The incident light raised the photocatalyst to an excited triplet state (PC*) that then transferred an electron to the oxime ethers with production of the radical anions 24 (Scheme 5). Loss of the stable phenolate type anions then occurred with release of the corresponding iminyl radicals that subsequently underwent 5-exo cyclisation and H-atom transfer with CHD to afford dihydropyrroles 25.
Furthermore, Leonori and co-workers reported recently that the dye Eosin Y (as PC) catalysed dihydropyrrole formation, simply with light of visible wavelength, when oxime ethers with O-2,4-dinitroaryl substitution 27 were employed as reactants [bib_ref] Visible-light-mediated generation of nitrogen-centered radicals: Metal-free hydroimination and iminohydroxylation cyclization reactions, Davies [/bib_ref]. Remarkably, Et 3 N could replace Eosin Y: visible light irradiation of 27 (EWG = 2,4-di-NO 2 ) with Et 3 N in CH 3 CN furnished imino-alcohols 31 in yields up to 85%. The complex of Et 3 N with the electron-poor ring of 27, on excitation with visible light, generated a radical anion that dissociated to give 2,4-dinitrophenoxide together with pyrrolidinylmethyl radical 28. The oxygen atom was believed to arise from an dihydropyrroles from O-aryl oxime ethers substituted with electron withdrawing groups (EWG) in their O-aryl units. Narasaka and co-workers prepared oxime ethers 23 containing 4-CN (or 2,4-di-NO2 or 4-CF3) aryl substituents. On inclusion of a catalytic amount of 1,5-dimethoxynaphthalene (DMN) and irradiation with UV light in 1,4-cyclohexadiene, dihydropyrroles 25 were isolated in good yields (Scheme 5) [bib_ref] Photochemical transformation of γ,δ-unsaturated ketone O-(p-cyanophenyl)oximes to 3,4-dihydro-2H-pyrrole derivatives, Mikami [/bib_ref]. The incident light raised the photocatalyst to an excited triplet state (PC*) that then transferred an electron to the oxime ethers with production of the radical anions 24 (Scheme 5). Loss of the stable phenolate type anions then occurred with release of the corresponding iminyl radicals that subsequently underwent 5-exo cyclisation and H-atom transfer with CHD to afford dihydropyrroles 25. Scheme 5. Photoredox catalytic routes from oxime ethers to dihydropyrroles [bib_ref] Visible-light-mediated generation of nitrogen-centered radicals: Metal-free hydroimination and iminohydroxylation cyclization reactions, Davies [/bib_ref] [bib_ref] Photochemical transformation of γ,δ-unsaturated ketone O-(p-cyanophenyl)oximes to 3,4-dihydro-2H-pyrrole derivatives, Mikami [/bib_ref]. Scheme 5. Photoredox catalytic routes from oxime ethers to dihydropyrroles [bib_ref] Visible-light-mediated generation of nitrogen-centered radicals: Metal-free hydroimination and iminohydroxylation cyclization reactions, Davies [/bib_ref] [bib_ref] Photochemical transformation of γ,δ-unsaturated ketone O-(p-cyanophenyl)oximes to 3,4-dihydro-2H-pyrrole derivatives, Mikami [/bib_ref].
Weinreb and co-workers described an alternative strategy in which oximes 31 could be used directly when treated with 2,6-dimethylbenzenesulfinyl chloride in the presence of Hunig's base (N,N-diisopropylethylamine, DIEA) and a radical trapping agent in dichloromethane (DCM) [bib_ref] Development of efficient new methodology for generation, cyclization and functional trapping of..., Lin [/bib_ref]. The best radical traps (Z) were found to be TEMPO and diphenyl diselenide; but CHD could also be used when in great excess (Scheme 6). Probably sulfinate esters 32 were first formed that dissociated to a caged iminyl/sulfonyl radical pair 33. The reformed N-sulfonylimines 34 then released iminyl radicals that cyclised and were trapped to afford functionalised dihydropyrroles 35 in moderate to good yields (Scheme 6). Furthermore, Leonori and co-workers reported recently that the dye Eosin Y (as PC) catalysed dihydropyrrole formation, simply with light of visible wavelength, when oxime ethers with O-2,4dinitroaryl substitution 27 were employed as reactants [bib_ref] Visible-light-mediated generation of nitrogen-centered radicals: Metal-free hydroimination and iminohydroxylation cyclization reactions, Davies [/bib_ref]. Remarkably, Et3N could replace Eosin Y: visible light irradiation of 27 (EWG = 2,4-di-NO2) with Et3N in CH3CN furnished imino-alcohols 31 in yields up to 85%. The complex of Et3N with the electron-poor ring of 27, on excitation with visible light, generated a radical anion that dissociated to give 2,4-dinitrophenoxide together with pyrrolidinylmethyl radical 28. The oxygen atom was believed to arise from an intermediate such as 29 that fragmented to nitrosophenoxide and a pyrrolidine-containing alkoxyl radical. The latter picked up an H-atom to deliver imino-alcohols 30 as the products (Scheme 5).
Weinreb and co-workers described an alternative strategy in which oximes 31 could be used directly when treated with 2,6-dimethylbenzenesulfinyl chloride in the presence of Hunig's base (N,N-diisopropylethylamine, DIEA) and a radical trapping agent in dichloromethane (DCM) [bib_ref] Development of efficient new methodology for generation, cyclization and functional trapping of..., Lin [/bib_ref]. The best radical traps (Z) were found to be TEMPO and diphenyl diselenide; but CHD could also be used when in great excess (Scheme 6). Probably sulfinate esters 32 were first formed that dissociated to a caged iminyl/sulfonyl radical pair 33. The reformed N-sulfonylimines 34 then released iminyl radicals that cyclised and were trapped to afford functionalised dihydropyrroles 35 in moderate to good yields (Scheme 6).
## Preparations of pyridine, quinoline, phenanthridine and related aza-arenes
Compounds containing the quinoline unit are hugely important to the well-being of society because they play essential roles across the areas of medicine, pharmacology, nutrition, dyes, and even electronics [bib_ref] Advances in the syntheses of quinoline and quinoline-annulated ring systems, Madapa [/bib_ref]. Bioactive natural products containing quinoline cores are widely distributed in many plants, marine plants, corals, sponges [bib_ref] Recent advances in research of natural and synthetic bioactive quinolines, Chung [/bib_ref] [bib_ref] Acetylcholinesterase and butyrylcholinesterase inhibitory activities of β-carboline and quinoline alkaloids derivatives from..., Zhao [/bib_ref] [bib_ref] Synthesis of quinolin-2-one alkaloid derivatives and their inhibitory activities against HIV-1 reverse..., Cheng [/bib_ref] [bib_ref] Quinoline alkaloids as intercalative topoisomerase inhibitors, Byler [/bib_ref] [bib_ref] Synthesis of the marine pyrroloiminoquinone alkaloids, discorhabdins, Wada [/bib_ref] and even in chestnut honey [bib_ref] Use of quinoline alkaloids as markers of the floral origin of chestnut..., Truchado [/bib_ref]. Much the same can be said for compounds containing phenanthridine units. They are noted for their ability to bind to DNA [bib_ref] Come-back of phenanthridine and phenanthridinium derivatives in the 21st century, Tumir [/bib_ref] and have antimicrobial, anti-inflammatory, and anti-tumor activities [bib_ref] Some aspects of the chemistry of quaternary benzo[c]phenanthridine alkaloids, Dostal [/bib_ref] [bib_ref] Quaternary benzo[c]phenanthridines sanguinarine and chelerythrine: A review of investigations from chemical and..., Dvorak [/bib_ref] [bib_ref] Slaninova, I. Benzo[c]phenanthridine alkaloids exhibit strong anti-proliferative activity in malignant melanoma cells..., Hammerova [/bib_ref] [bib_ref] Antitumour activities of sanguinarine and related alkaloids, Slaninova [/bib_ref] [bib_ref] Pseudocyanides of sanguinarine and chelerythrine and their series of structurally simple analogues..., Cao [/bib_ref]. Because of this importance to the field of drug development, synthetic methods for molecules containing these structural units, particularly short, mild and atom-efficient ones, have attracted great attention.
## Preparations of aza-arenes from carbonyl oximes
Iminyl radicals 36 containing suitably sited aromatic acceptor units usually ring close in 6-endo mode with formation of a 6-membered N-containing ring 38 (Scheme 7). This rule holds good for cyclisations onto a wide range of aromatic acceptors including benzene, naphthalene, furan, thiophene,
## Preparations of pyridine, quinoline, phenanthridine and related aza-arenes
Compounds containing the quinoline unit are hugely important to the well-being of society because they play essential roles across the areas of medicine, pharmacology, nutrition, dyes, and even electronics [bib_ref] Advances in the syntheses of quinoline and quinoline-annulated ring systems, Madapa [/bib_ref]. Bioactive natural products containing quinoline cores are widely distributed in many plants, marine plants, corals, sponges [bib_ref] Recent advances in research of natural and synthetic bioactive quinolines, Chung [/bib_ref] [bib_ref] Acetylcholinesterase and butyrylcholinesterase inhibitory activities of β-carboline and quinoline alkaloids derivatives from..., Zhao [/bib_ref] [bib_ref] Synthesis of quinolin-2-one alkaloid derivatives and their inhibitory activities against HIV-1 reverse..., Cheng [/bib_ref] [bib_ref] Quinoline alkaloids as intercalative topoisomerase inhibitors, Byler [/bib_ref] [bib_ref] Synthesis of the marine pyrroloiminoquinone alkaloids, discorhabdins, Wada [/bib_ref] and even in chestnut honey [bib_ref] Use of quinoline alkaloids as markers of the floral origin of chestnut..., Truchado [/bib_ref]. Much the same can be said for compounds containing phenanthridine units. They are noted for their ability to bind to DNA [bib_ref] Come-back of phenanthridine and phenanthridinium derivatives in the 21st century, Tumir [/bib_ref] and have antimicrobial, anti-inflammatory, and anti-tumor activities [bib_ref] Some aspects of the chemistry of quaternary benzo[c]phenanthridine alkaloids, Dostal [/bib_ref] [bib_ref] Quaternary benzo[c]phenanthridines sanguinarine and chelerythrine: A review of investigations from chemical and..., Dvorak [/bib_ref] [bib_ref] Slaninova, I. Benzo[c]phenanthridine alkaloids exhibit strong anti-proliferative activity in malignant melanoma cells..., Hammerova [/bib_ref] [bib_ref] Antitumour activities of sanguinarine and related alkaloids, Slaninova [/bib_ref] [bib_ref] Pseudocyanides of sanguinarine and chelerythrine and their series of structurally simple analogues..., Cao [/bib_ref]. Because of this importance to the field of drug development, synthetic methods for molecules containing these structural units, particularly short, mild and atom-efficient ones, have attracted great attention.
## Preparations of aza-arenes from carbonyl oximes
Iminyl radicals 36 containing suitably sited aromatic acceptor units usually ring close in 6-endo mode with formation of a 6-membered N-containing ring 38 (Scheme 7). This rule holds good for cyclisations onto a wide range of aromatic acceptors including benzene, naphthalene, furan, thiophene, indole, pyridine and analogous moieties. As would be expected for less-favoured 6-endo processes, the rate constants for these iminyl ring closures are significantly smaller than for 5-exo cyclisations [bib_ref] Interplay of ortho-with spiro-cyclisation during iminyl radical closures onto arenes and heteroarenes, Mcburney [/bib_ref]. Never-the-less practicable preparative protocols have been established for many mono-and poly-cyclic heterocycles. restoration of aromaticity to the acceptor ring has usually been observed as in quinoline derivative 41. This can result when some radical in the system abstracts the highly labile tertiary H-atom of the cyclohexadienyl ring in radical 38. However, this is a disproportionation that requires two radicals to meet and so is not favored because reactive radical concentrations are always very low. The alternative is that an acceptor molecule A is either added, or is adventitiously present, and single electron transfer (SET) occurs with production of the corresponding cyclohexadienyl cations 40. These rapidly deprotonate to yield cyclised products 41 with restored aromaticity. This oxidative process is facilitated by non-H-atom donor solvents such as PhCF3 or t-BuOH. Scheme 7. Iminyl radical 6-endo ring closures onto aromatic acceptors.
Formyl and acyl derivatives of biphenyl, 4-phenylpyridine, 2-phenylthiophene, 3-phenylpyrazole, 2-phenylnaphthalene, 2-phenylindole and analogous aromatics are readily accessible and can be converted to the corresponding acyl oximes without difficulty (see . UV photolyses of these precursors through Pyrex in acetonitrile or t-butanol solutions enabled several 6-substituted phenanthridines 42 to be prepared [bib_ref] New light-induced iminyl radical cyclization reactions of acyloximes to isoquinolines, Alonso [/bib_ref] [bib_ref] Photochemistry of acyloximes: Synthesis of heterocycles and natural products, Alonso [/bib_ref]. Similarly, benzo[c] [bib_ref] The chemical space project, Reymond [/bib_ref] [bib_ref] Quest for the rings. In silico exploration of ring universe to identify..., Ertl [/bib_ref] naphthyridines 43, and thieno[3,2c]isoquinolines 44 were conveniently prepared from the corresponding acyloximes. Scheme 8. Preparations of tri-and tetra-cyclic heterocycles from acyl oximes [bib_ref] New light-induced iminyl radical cyclization reactions of acyloximes to isoquinolines, Alonso [/bib_ref] [bib_ref] Photochemistry of acyloximes: Synthesis of heterocycles and natural products, Alonso [/bib_ref]. Analogous photochemical preparations were described for 2,4-dimethyl-2H-pyrazolo[4,3-c]quinoline 45 and for benzo[i]phenanthridine 46. However, the aldoxime precursor from 2-phenylindole gave only 2-phenyl-1H-indole-3-carbonitrile 47 in low yield. Isoquinoline derivatives were also obtained in good yields from photolyses of mixtures of benzophenone acetyl oxime with alkynes in t-butanol [bib_ref] New light-induced iminyl radical cyclization reactions of acyloximes to isoquinolines, Alonso [/bib_ref].
Huang, Deng and co-workers recently described a novel method of making 2-arylpyridines by treatment of O-acyl oxime esters and acroleins with iodine and triethylamine in toluene at 120 °C [bib_ref] Metal-free assembly of polysubstituted pyridines from oximes and acroleins, Huang [/bib_ref]. The method had wide scope and yields ranged from moderate to excellent. It is usually necessary to work with precursors having the second imine substituent R 1 either Me or Ar. If R 1 is H (or a branched alkyl group) formation of a nitrile 37 is either the main process, or 37 is an important by-product (Scheme 7). Nitrile formation may entail an electrocyclic process of the oxime ester; in which case the proportion may be diminished by suitable solvent choice. With several types of oxime esters cyclisations onto aromatic rings initially create cyclohexadienyl type radicals 38. If a suitable H-donor is present as solvent, or otherwise, H-atom transfer to radicals 38 yields cyclohexadienes 39 as a mixture of isomers. In practice this route has rarely been developed. Instead restoration of aromaticity to the acceptor ring has usually been observed as in quinoline derivative 41. This can result when some radical in the system abstracts the highly labile tertiary H-atom of the cyclohexadienyl ring in radical 38. However, this is a disproportionation that requires two radicals to meet and so is not favored because reactive radical concentrations are always very low. The alternative is that an acceptor molecule A is either added, or is adventitiously present, and single electron transfer (SET) occurs with production of the corresponding cyclohexadienyl cations 40. These rapidly deprotonate to yield cyclised products 41 with restored aromaticity. This oxidative process is facilitated by non-H-atom donor solvents such as PhCF 3 or t-BuOH.
Formyl and acyl derivatives of biphenyl, 4-phenylpyridine, 2-phenylthiophene, 3-phenylpyrazole, 2-phenylnaphthalene, 2-phenylindole and analogous aromatics are readily accessible and can be converted to the corresponding acyl oximes without difficulty (see . UV photolyses of these precursors through Pyrex in acetonitrile or t-butanol solutions enabled several 6-substituted phenanthridines 42 to be prepared [bib_ref] New light-induced iminyl radical cyclization reactions of acyloximes to isoquinolines, Alonso [/bib_ref] [bib_ref] Photochemistry of acyloximes: Synthesis of heterocycles and natural products, Alonso [/bib_ref]. Similarly, benzo[c] [bib_ref] The chemical space project, Reymond [/bib_ref] [bib_ref] Quest for the rings. In silico exploration of ring universe to identify..., Ertl [/bib_ref] naphthyridines 43, and thieno[3,2-c] isoquinolines 44 were conveniently prepared from the corresponding acyloximes.
Analogous photochemical preparations were described for 2,4-dimethyl-2H-pyrazolo[4,3-c]quinoline 45 and for benzo[i]phenanthridine 46. However, the aldoxime precursor from 2-phenylindole gave only 2-phenyl-1H-indole-3-carbonitrile 47 in low yield. Isoquinoline derivatives were also obtained in good yields from photolyses of mixtures of benzophenone acetyl oxime with alkynes in t-butanol [bib_ref] New light-induced iminyl radical cyclization reactions of acyloximes to isoquinolines, Alonso [/bib_ref].
Formyl and acyl derivatives of biphenyl, 4-phenylpyridine, 2-phenylthiophene, 3-phenylpyrazole, 2-phenylnaphthalene, 2-phenylindole and analogous aromatics are readily accessible and can be converted to the corresponding acyl oximes without difficulty (see . UV photolyses of these precursors through Pyrex in acetonitrile or t-butanol solutions enabled several 6-substituted phenanthridines 42 to be prepared [bib_ref] New light-induced iminyl radical cyclization reactions of acyloximes to isoquinolines, Alonso [/bib_ref] [bib_ref] Photochemistry of acyloximes: Synthesis of heterocycles and natural products, Alonso [/bib_ref]. Similarly, benzo[c] [bib_ref] The chemical space project, Reymond [/bib_ref] [bib_ref] Quest for the rings. In silico exploration of ring universe to identify..., Ertl [/bib_ref] naphthyridines 43, and thieno[3,2c]isoquinolines 44 were conveniently prepared from the corresponding acyloximes.
## Scheme 8.
Preparations of tri-and tetra-cyclic heterocycles from acyl oximes [bib_ref] New light-induced iminyl radical cyclization reactions of acyloximes to isoquinolines, Alonso [/bib_ref] [bib_ref] Photochemistry of acyloximes: Synthesis of heterocycles and natural products, Alonso [/bib_ref]. Analogous photochemical preparations were described for 2,4-dimethyl-2H-pyrazolo[4,3-c]quinoline 45 and for benzo[i]phenanthridine 46. However, the aldoxime precursor from 2-phenylindole gave only 2-phenyl-1H-indole-3-carbonitrile 47 in low yield. Isoquinoline derivatives were also obtained in good yields from photolyses of mixtures of benzophenone acetyl oxime with alkynes in t-butanol [bib_ref] New light-induced iminyl radical cyclization reactions of acyloximes to isoquinolines, Alonso [/bib_ref].
Huang, Deng and co-workers recently described a novel method of making 2-arylpyridines by treatment of O-acyl oxime esters and acroleins with iodine and triethylamine in toluene at 120 °C [bib_ref] Metal-free assembly of polysubstituted pyridines from oximes and acroleins, Huang [/bib_ref]. The method had wide scope and yields ranged from moderate to excellent. Scheme 8. Preparations of tri-and tetra-cyclic heterocycles from acyl oximes [bib_ref] New light-induced iminyl radical cyclization reactions of acyloximes to isoquinolines, Alonso [/bib_ref] [bib_ref] Photochemistry of acyloximes: Synthesis of heterocycles and natural products, Alonso [/bib_ref].
Huang, Deng and co-workers recently described a novel method of making 2-arylpyridines by treatment of O-acyl oxime esters and acroleins with iodine and triethylamine in toluene at 120˝C [bib_ref] Metal-free assembly of polysubstituted pyridines from oximes and acroleins, Huang [/bib_ref]. The method had wide scope and yields ranged from moderate to excellent.
Symmetrical dioxime oxalates containing aromatic acceptors 48 were prepared in essentially quantitative yields, via the oxime oxalyl chlorides, from the corresponding oximes. On UV photolyses at ambient temperature in MeCN solution these returned phenanthridines 49 in clean and atom-efficient processes [bib_ref] Dioxime oxalates; new iminyl radical precursors for syntheses of N-heterocycles, Portela-Cubillo [/bib_ref]. Best yields were obtained on inclusion of MAP (ArMeC=O) and it seems this acted as both a photosensitizer and as an electron acceptor to facilitate the final re-aromatization (see . Symmetrical dioxime oxalates containing aromatic acceptors 48 were prepared in essentially quantitative yields, via the oxime oxalyl chlorides, from the corresponding oximes. On UV photolyses at ambient temperature in MeCN solution these returned phenanthridines 49 in clean and atom-efficient processes [bib_ref] Dioxime oxalates; new iminyl radical precursors for syntheses of N-heterocycles, Portela-Cubillo [/bib_ref]. Best yields were obtained on inclusion of MAP (ArMeC=O) and it seems this acted as both a photosensitizer and as an electron acceptor to facilitate the final re-aromatization (see Scheme 9). Scheme 9. Preparation of phenanthridines from dioxime oxalates [bib_ref] Dioxime oxalates; new iminyl radical precursors for syntheses of N-heterocycles, Portela-Cubillo [/bib_ref].
Oxime carbonates 52 can be prepared either by treatment of an oxime with a chloroformate 50 or via the 1H-imidazole-1-carboxylates 51. The latter can be obtained from reaction of an alcohol with carbonyldiimidazole (CDI) (see . For the preparation of aza-arenes commercial ethyl chloroformate (50, R 2 = Et) was found to be very convenient because the main by-product (EtOH) is volatile and easily separated. UV photolyses of appropriately functionalised oxime carbonates, with MAP as additive, enabled phenanthridines 53, methylfuro-and methylthieno-[2,3-c]quinolines 54 to be prepared as well as the 5-methylbenzofuro-and 5-methylbenzo [bib_ref] Experimental and computational approaches to estimate solubility and permeability in drug discovery..., Lipinski [/bib_ref] [bib_ref] The art and practice of structure-based drug design: A molecular modelling perspective, Bohacek [/bib_ref] thieno-[3,2-c]isoquinolines 55 in generally good yields [bib_ref] Interplay of ortho-with spiro-cyclisation during iminyl radical closures onto arenes and heteroarenes, Mcburney [/bib_ref] [bib_ref] UV promoted phenanthridine syntheses from oxime carbonate derived iminyl radicals, Mcburney [/bib_ref]. Scheme 9. Preparation of phenanthridines from dioxime oxalates [bib_ref] Dioxime oxalates; new iminyl radical precursors for syntheses of N-heterocycles, Portela-Cubillo [/bib_ref].
Oxime carbonates 52 can be prepared either by treatment of an oxime with a chloroformate 50 or via the 1H-imidazole-1-carboxylates 51. The latter can be obtained from reaction of an alcohol with carbonyldiimidazole (CDI) (see . For the preparation of aza-arenes commercial ethyl chloroformate (50, R 2 = Et) was found to be very convenient because the main by-product (EtOH) is volatile and easily separated. UV photolyses of appropriately functionalised oxime carbonates, with MAP as additive, enabled phenanthridines 53, methylfuro-and methylthieno-[2,3-c]quinolines 54 to be prepared as well as the 5-methylbenzofuro-and 5-methylbenzo [bib_ref] Experimental and computational approaches to estimate solubility and permeability in drug discovery..., Lipinski [/bib_ref] [bib_ref] The art and practice of structure-based drug design: A molecular modelling perspective, Bohacek [/bib_ref] thieno-[3,2-c]isoquinolines 55 in generally good yields [bib_ref] Interplay of ortho-with spiro-cyclisation during iminyl radical closures onto arenes and heteroarenes, Mcburney [/bib_ref] [bib_ref] UV promoted phenanthridine syntheses from oxime carbonate derived iminyl radicals, Mcburney [/bib_ref].
Oxime carbamates 56 offer another alternative for aza-arene syntheses but have not yet been much exploited. The Et 2 N aminyl group is sufficiently small that by-products derived from it volatilize away. UV irradiation of 56 in PhCF 3 at room temperature and with MAP additive provided 3-methoxy-6-methylphenanthridine 57 in 60% yield (Scheme 11) [bib_ref] Dissociation or cyclization: Options for a triad of radicals released from oxime..., Mcburney [/bib_ref]. A slightly higher yield (61%) was obtained in the absence of MAP. This was a worthwhile finding because residues of the photosensitizer can be troublesome to remove. carbonyldiimidazole (CDI) (see . For the preparation of aza-arenes commercial ethyl chloroformate (50, R 2 = Et) was found to be very convenient because the main by-product (EtOH) is volatile and easily separated. UV photolyses of appropriately functionalised oxime carbonates, with MAP as additive, enabled phenanthridines 53, methylfuro-and methylthieno-[2,3-c]quinolines 54 to be prepared as well as the 5-methylbenzofuro-and 5-methylbenzo [bib_ref] Experimental and computational approaches to estimate solubility and permeability in drug discovery..., Lipinski [/bib_ref] [bib_ref] The art and practice of structure-based drug design: A molecular modelling perspective, Bohacek [/bib_ref] thieno-[3,2-c]isoquinolines 55 in generally good yields [bib_ref] Interplay of ortho-with spiro-cyclisation during iminyl radical closures onto arenes and heteroarenes, Mcburney [/bib_ref] [bib_ref] UV promoted phenanthridine syntheses from oxime carbonate derived iminyl radicals, Mcburney [/bib_ref]. Scheme 10. Preparations of aza-arenes from oxime carbonates [bib_ref] Interplay of ortho-with spiro-cyclisation during iminyl radical closures onto arenes and heteroarenes, Mcburney [/bib_ref] [bib_ref] UV promoted phenanthridine syntheses from oxime carbonate derived iminyl radicals, Mcburney [/bib_ref].
Oxime carbamates 56 offer another alternative for aza-arene syntheses but have not yet been much exploited. The Et2N aminyl group is sufficiently small that by-products derived from it volatilize away. UV irradiation of 56 in PhCF3 at room temperature and with MAP additive provided 3-methoxy-6methylphenanthridine 57 in 60% yield (Scheme 11) [bib_ref] Dissociation or cyclization: Options for a triad of radicals released from oxime..., Mcburney [/bib_ref]. A slightly higher yield (61%) was obtained in the absence of MAP. This was a worthwhile finding because residues of the photosensitizer can be troublesome to remove. Scheme 10. Preparations of aza-arenes from oxime carbonates [bib_ref] Interplay of ortho-with spiro-cyclisation during iminyl radical closures onto arenes and heteroarenes, Mcburney [/bib_ref] [bib_ref] UV promoted phenanthridine syntheses from oxime carbonate derived iminyl radicals, Mcburney [/bib_ref].
## Preparations of aza-arenes from oxime ethers
As mentioned above (Section 2.2) O-aryl oxime ethers, including those with appropriately placed aryl acceptor units, can easily be made from carbonyl compounds. The 3-phenylpropanone O-phenyl oxime ethers 58 were subjected to microwave irradiation at 160 °C in t-BuPh together with emimPF6 ionic liquid. Quinoline 59a and tetrahydroacridine 59b were thereby produced in moderate yields [bib_ref] Microwave-assisted preparations of dihydropyrroles from alkenone O-phenyl oximes, Portela-Cubillo [/bib_ref] [bib_ref] Microwave-assisted syntheses of N-heterocycles using alkenone-, alkynone-and aryl-carbonyl O-phenyl oximes: Formal synthesis..., Portela-Cubillo [/bib_ref] (Scheme 12).
## Scheme 12.
Microwave promoted preparations of aza-arenes from oxime ethers [bib_ref] Microwave-assisted preparations of dihydropyrroles from alkenone O-phenyl oximes, Portela-Cubillo [/bib_ref] [bib_ref] Microwave-assisted syntheses of N-heterocycles using alkenone-, alkynone-and aryl-carbonyl O-phenyl oximes: Formal synthesis..., Portela-Cubillo [/bib_ref]
## Preparations of aza-arenes from oxime ethers
As mentioned above (Section 2.2) O-aryl oxime ethers, including those with appropriately placed aryl acceptor units, can easily be made from carbonyl compounds. The 3-phenylpropanone O-phenyl oxime ethers 58 were subjected to microwave irradiation at 160˝C in t-BuPh together with emimPF 6 ionic liquid. Quinoline 59a and tetrahydroacridine 59b were thereby produced in moderate yields [bib_ref] Microwave-assisted preparations of dihydropyrroles from alkenone O-phenyl oximes, Portela-Cubillo [/bib_ref] [bib_ref] Microwave-assisted syntheses of N-heterocycles using alkenone-, alkynone-and aryl-carbonyl O-phenyl oximes: Formal synthesis..., Portela-Cubillo [/bib_ref]
## Preparations of aza-arenes from oxime ethers
As mentioned above (Section 2.2) O-aryl oxime ethers, including those with appropriately placed aryl acceptor units, can easily be made from carbonyl compounds. The 3-phenylpropanone O-phenyl oxime ethers 58 were subjected to microwave irradiation at 160 °C in t-BuPh together with emimPF6 ionic liquid. Quinoline 59a and tetrahydroacridine 59b were thereby produced in moderate yields [bib_ref] Microwave-assisted preparations of dihydropyrroles from alkenone O-phenyl oximes, Portela-Cubillo [/bib_ref] [bib_ref] Microwave-assisted syntheses of N-heterocycles using alkenone-, alkynone-and aryl-carbonyl O-phenyl oximes: Formal synthesis..., Portela-Cubillo [/bib_ref] (Scheme 12). Similarly, biphenyl-2-carbaldehyde O-phenyl oximes 60a and 2-arylnicotinaldehyde O-phenyl oximes 60b, with either electron releasing or electron withdrawing substituents, yielded the corresponding phenanthridine 61a or benzo[h] [bib_ref] The chemical space project, Reymond [/bib_ref] [bib_ref] Self-organization of matter and the evolution of biological macromolecules, Eigen [/bib_ref] naphthyridine derivatives 61b respectively. The scope of the method was easily extended such that benzo[k]phenanthridine 63 and benzo[b]thieno[2,3-c]quinoline 65 were prepared in a very few steps and in acceptable yields (Scheme 12).
## Photoredox catalyzed preparations of aza-arenes from oxime derivatives
The advantages of photoredox catalyzed processes include that visible light, rather than more damaging UV radiation, is usually sufficient and that only catalytic quantities of the PC are required. Several such methods have been reported recently for aza-arene preparations with oxime derivatives as starting materials For example, Zhang, Yu and co-workers generated iminyl radicals from a wide range of aroyl oximes with fac-[Ir(ppy) 3 ] (ppy = 2-phenylpyridine) as PC [bib_ref] Visible-light-promoted iminyl-radical formation from acyl oximes: A unified approach to pyridines, quinolines,..., Jiang [/bib_ref]. The most reliable and efficient aroyl group was shown to be 4-trifluoromethylbenzoyl; although other electron deficient benzoates succeeded to variable extents. Visible light irradiations of biphenyl aroyl oximes 66 with catalytic quantities of fac-[Ir(ppy) 3 ] in DMF produced phenanthridine derivatives 67 in very impressive yields (Scheme 13). The PC catalysed the formation of a radical anion from the substrate that then released an iminyl radical together with the 4-trifluorobenzoate anion. The non-H-atom donor solvent DMF favoured production of the fully aromatic products shown. Using similar methodology, the precursors 68 yielded quinolines 69 with a good range of functionality. Furthermore, aroyl oxime esters 70, with butadienyl substituents, led to iminyl radicals that ring closed selectively in 6-endo mode with eventual production of pyridine derivatives 71, generally in very good yields. Yu and An developed a convenient one-pot tactic that enabled aza-arenes to be prepared in one step from readily available carbonyl compounds [bib_ref] Visible-light-promoted and one-pot synthesis of phenanthridines and quinolines from aldehydes and O-acyl..., An [/bib_ref]. A somewhat related method was reported by de Lijser and co-workers who employed 2′-arylbenzaldehyde oxime ethers 76 together with 9,10-dicyanoanthracene (DCA) [bib_ref] Catalytic oxidative cyclization of 2 1 -arylbenzaldehyde oxime ethers under photoinduced electron..., Hofstra [/bib_ref]. Photolyses at 420 nm in CD3CN solutions enabled a set of substituted phenanthridines 80 to be obtained. This process was believed to proceed via radical cation 77 that ring closed by nucleophilic attack of the aromatic unit onto the N-atom of the oxime ether 78 (Scheme 14). Subsequent loss of methoxyl radicals led to cyclohexadienyl cations 79 that underwent deprotonation to afford the product phenanthridines. Yields were variable with strongly electron-releasing substituents Y being deleterious. The main by-Scheme 13. Photoredox methods for preparations of aza-arenes from oxime derivatives [bib_ref] Visible-light-promoted iminyl-radical formation from acyl oximes: A unified approach to pyridines, quinolines,..., Jiang [/bib_ref] [bib_ref] Visible-light-promoted and one-pot synthesis of phenanthridines and quinolines from aldehydes and O-acyl..., An [/bib_ref].
Yu and An developed a convenient one-pot tactic that enabled aza-arenes to be prepared in one step from readily available carbonyl compounds [bib_ref] Visible-light-promoted and one-pot synthesis of phenanthridines and quinolines from aldehydes and O-acyl..., An [/bib_ref]. A somewhat related method was reported by de Lijser and co-workers who employed 2 1 -arylbenzaldehyde oxime ethers 76 together with 9,10-dicyanoanthracene (DCA) [bib_ref] Catalytic oxidative cyclization of 2 1 -arylbenzaldehyde oxime ethers under photoinduced electron..., Hofstra [/bib_ref]. Photolyses at 420 nm in CD 3 CN solutions enabled a set of substituted phenanthridines 80 to be obtained. This process was believed to proceed via radical cation 77 that ring closed by nucleophilic attack of the aromatic unit onto the N-atom of the oxime ether 78 (Scheme 14). Subsequent loss of methoxyl radicals led to cyclohexadienyl cations 79 that underwent deprotonation to afford the product phenanthridines. Yields were variable with strongly electron-releasing substituents Y being deleterious. The main by-product was the volatile and innocuous MeOH and no nitrile formation was observed.
Yu and An developed a convenient one-pot tactic that enabled aza-arenes to be prepared in one step from readily available carbonyl compounds [bib_ref] Visible-light-promoted and one-pot synthesis of phenanthridines and quinolines from aldehydes and O-acyl..., An [/bib_ref]. They used O-(4-cyanobenzoyl)hydroxylamine [ArC(O)ONH2] in conjunction with biphenyl aldehydes 72. To promote aroyl oxime formation 4-chlorobenzenesulfonic acid (CBSA) was employed as additive. On irradiation with visible light from white LED strips, phenanthridines 74 were obtained directly without isolation of the intermediate oxime esters 73. A similar one-pot procedure, but employing blue LED strip illumination, was established for preparations of quinoline derivatives 75 from cinnamaldehyde type precursors.
A somewhat related method was reported by de Lijser and co-workers who employed 2′-arylbenzaldehyde oxime ethers 76 together with 9,10-dicyanoanthracene (DCA) [bib_ref] Catalytic oxidative cyclization of 2 1 -arylbenzaldehyde oxime ethers under photoinduced electron..., Hofstra [/bib_ref]. Photolyses at 420 nm in CD3CN solutions enabled a set of substituted phenanthridines 80 to be obtained. This process was believed to proceed via radical cation 77 that ring closed by nucleophilic attack of the aromatic unit onto the N-atom of the oxime ether 78 (Scheme 14). Subsequent loss of methoxyl radicals led to cyclohexadienyl cations 79 that underwent deprotonation to afford the product phenanthridines. Yields were variable with strongly electron-releasing substituents Y being deleterious. The main byproduct was the volatile and innocuous MeOH and no nitrile formation was observed.
## Scheme 14.
Photoredox production of phenanthridines from 2′-arylbiphenyl oxime ethers [bib_ref] Catalytic oxidative cyclization of 2 1 -arylbenzaldehyde oxime ethers under photoinduced electron..., Hofstra [/bib_ref].
## Preparations of quinazoline, quinoxaline and related heterocycles from oxime derivatives
Bioactivities of various kinds have been associated with numerous substances having structures incorporating quinazoline rings. A number are enzyme inhibitors [bib_ref] A specific inhibitor of the epidermal growth factor receptor tyrosine kinase, Fry [/bib_ref] , some display antiviral or anticancer activity [bib_ref] Quinazoline derivatives with antitubercular activity, Kunes [/bib_ref] [bib_ref] Pharmacological rescue of mutant p53 conformation and function, Foster [/bib_ref] ; others are antibacterial agents [bib_ref] Synthesis and biological activity of novel antibacterial quinazolines, Bedi [/bib_ref]. The growing library of approved drugs containing this structural unit, such as erlotinib (Tarceva ® ) [bib_ref] Discovery of novel small-molecule inhibitors of human epidermal growth factor receptor-2: Combined..., Gundla [/bib_ref] , prazosin (Minipress ® , Vasoflex ® , Lentopres ® , Hypovase ® [bib_ref] Molecular features of the prazosin molecule required for activation of transport-P, Mendes Da Silva [/bib_ref] and gefitinib (Iressa ® ) [bib_ref] Synthesis and evaluation of fused tricyclic quinazoline analogs as ATP site inhibitors..., Rewcastle [/bib_ref] [bib_ref] Syntheses of 4-(indol-3-yl)quinazolines-A new class of epidermal growth factor receptor tyrosine kinase..., Luth [/bib_ref] , demonstrates the transferable character of aspects of its properties. Steady progress has been made in the development of synthetic methods for quinazoline alkaloids in the last few decades [bib_ref] Synthesis of quinazolinones and quinazolines, Connolly [/bib_ref] [bib_ref] Microwave-assisted synthesis of bioactive quinazolines and quinazolinones, Besson [/bib_ref]. Some useful microwave promoted procedures have also been described including those based on: aniline N-ethyl carbamates [bib_ref] A microwave improvement in the synthesis of the quinazoline scaffold, Chilin [/bib_ref] , cyano-aromatic Scheme 14. Photoredox production of phenanthridines from 2 1 -arylbiphenyl oxime ethers [bib_ref] Catalytic oxidative cyclization of 2 1 -arylbenzaldehyde oxime ethers under photoinduced electron..., Hofstra [/bib_ref].
## Preparations of quinazoline, quinoxaline and related heterocycles from oxime derivatives
Bioactivities of various kinds have been associated with numerous substances having structures incorporating quinazoline rings. A number are enzyme inhibitors [bib_ref] A specific inhibitor of the epidermal growth factor receptor tyrosine kinase, Fry [/bib_ref] , some display antiviral or anticancer activity [bib_ref] Quinazoline derivatives with antitubercular activity, Kunes [/bib_ref] [bib_ref] Pharmacological rescue of mutant p53 conformation and function, Foster [/bib_ref] ; others are antibacterial agents [bib_ref] Synthesis and biological activity of novel antibacterial quinazolines, Bedi [/bib_ref]. The growing library of approved drugs containing this structural unit, such as erlotinib (Tarceva ® ) [bib_ref] Discovery of novel small-molecule inhibitors of human epidermal growth factor receptor-2: Combined..., Gundla [/bib_ref] , prazosin (Minipress ® , Vasoflex ® , Lentopres ® , Hypovase ® [bib_ref] Molecular features of the prazosin molecule required for activation of transport-P, Mendes Da Silva [/bib_ref] and gefitinib (Iressa ® ) [bib_ref] Synthesis and evaluation of fused tricyclic quinazoline analogs as ATP site inhibitors..., Rewcastle [/bib_ref] [bib_ref] Syntheses of 4-(indol-3-yl)quinazolines-A new class of epidermal growth factor receptor tyrosine kinase..., Luth [/bib_ref] , demonstrates the transferable character of aspects of its properties. Steady progress has been made in the development of synthetic methods for quinazoline alkaloids in the last few decades [bib_ref] Synthesis of quinazolinones and quinazolines, Connolly [/bib_ref] [bib_ref] Microwave-assisted synthesis of bioactive quinazolines and quinazolinones, Besson [/bib_ref]. Some useful microwave promoted procedures have also been described including those based on: aniline N-ethyl carbamates [bib_ref] A microwave improvement in the synthesis of the quinazoline scaffold, Chilin [/bib_ref] , cyano-aromatic compounds with anthranilonitrile [bib_ref] Microwave-enhanced synthesis of 4-aminoquinazolines, Seijas [/bib_ref] , N-(2-cyanophenyl)-N,N-dimethylformamidine derivatives [bib_ref] Efficient synthesis of 4-aminoquinazoline and thieno[3,2-d]pyrimidin-4-ylamine derivatives by microwave irradiation, Yoon [/bib_ref] , N-arylamidines with various aldehydes [bib_ref] A catalyst-and solvent-free selective approach to biologically important quinazolines and benzo[g]quinazoline, Kumar [/bib_ref] and 2-aminoaryl imines with aldehydes [bib_ref] Regioselective addition of Grignard reagents to 2,6-dicyanoanilines and cyclization to new quinazoline..., Maitraie [/bib_ref]. The common methods of making the quinazoline ring involve multi-step precursor preparations or special reagents. A pleasing and effective exception is the CuCl 2 -catalysed reaction of aldehydes with anthranilamide [bib_ref] A novel method for the synthesis of 4(3H)-quinazolinones, Abdel-Jalil [/bib_ref].
## Preparations of quinazoline derivatives
Aminoarylalkanone O-phenyl oximes 81 can be efficiently prepared from the corresponding ketones (Scheme 15). On mixing these starting materials with an aldehyde and microwave irradiating at 160˝C, in toluene as H-donor solvent, with emimPF 6 as ionic liquid, imine intermediates 82 were formed. Dissociation to phenoxyl and iminyl radicals 83 took place in one pot, without the need to isolate the imine intermediates 82. Ring closure was exclusively in the 6-endo mode with production of aminyl radicals 84 that were reduced to 1,2-dihydroquinazolines 85 by H-atom donation from the solvent [bib_ref] 2-(Aminoaryl)alkanone O-phenyl oximes: Versatile reagents for syntheses of quinazolines, Portela-Cubillo [/bib_ref] [bib_ref] Microwave-promoted syntheses of quinazolines and dihydroquinazolines from 2-aminoarylalkanone O-phenyl oximes, Portela-Cubillo [/bib_ref].
This proved to be a rapid, mild, reproducible and efficient process with aliphatic, aromatic and heterocyclic aldehydes; the only by-product being phenol. Analogous methodology with 81 and ketones was not successful, except with a few ketones such as cyclohexanone. The dihydroquinazolines 85 slowly oxidized to the corresponding quinazolines in air or oxygen. Alternatively, they could be converted to the latter in high yields by µwave irradiation with DDQ (2,3-dichloro-5, 6-dicyanobenzoquinone) at 100˝C in dichloromethane solvent. ketones (Scheme 15). On mixing these starting materials with an aldehyde and microwave irradiating at 160 °C, in toluene as H-donor solvent, with emimPF6 as ionic liquid, imine intermediates 82 were formed. Dissociation to phenoxyl and iminyl radicals 83 took place in one pot, without the need to isolate the imine intermediates 82. Ring closure was exclusively in the 6-endo mode with production of aminyl radicals 84 that were reduced to 1,2-dihydroquinazolines 85 by H-atom donation from the solvent [bib_ref] 2-(Aminoaryl)alkanone O-phenyl oximes: Versatile reagents for syntheses of quinazolines, Portela-Cubillo [/bib_ref] [bib_ref] Microwave-promoted syntheses of quinazolines and dihydroquinazolines from 2-aminoarylalkanone O-phenyl oximes, Portela-Cubillo [/bib_ref] (Scheme 15). This proved to be a rapid, mild, reproducible and efficient process with aliphatic, aromatic and heterocyclic aldehydes; the only by-product being phenol. Analogous methodology with 81 and ketones was not successful, except with a few ketones such as cyclohexanone. The dihydroquinazolines 85 slowly oxidized to the corresponding quinazolines in air or oxygen. Alternatively, they could be converted to the latter in high yields by μwave irradiation with DDQ (2,3-dichloro-5,6dicyanobenzoquinone) at 100 °C in dichloromethane solvent.
Inclusion of ZnCl2, a known promoter of imine formation [bib_ref] First synthesis of marine alkaloid (˘)-bengacarboline, Pouilhes [/bib_ref] , in practice led directly to the corresponding fully aromatic quinazolines 87. Good to excellent yields were obtained with aliphatic aldehydes, aromatic aldehydes having either electron-withdrawing or electron releasing substituents and with heterocyclic aldehydes. The method was also tolerant of substituents in the aminoaryl rings. It's likely that, in addition to encouraging imine formation, the ZnCl2 forms a complex with the iminyl N-atom in radical cation 86. The acidity of the H-atom adjacent to the cation would thereby be increased, thus facilitating proton-coupled electron transfer (PCET) and thence to the quinazolines 87 (Scheme 15).
Conventional organic preparations of quinazolinones usually start with derivatives of anthranilic acid [bib_ref] Synthesis of quinazolinones and quinazolines, Connolly [/bib_ref] [bib_ref] Synthesis of 2,3-disubstituted-quinazolin-4-(3H)-ones, Patil [/bib_ref]. Several good Pd-and Cu-catalyzed methods for compounds containing this structure have also been published [bib_ref] Synthesis of 3-substituted and 2,3-disubstituted quinazolinones via Cu-catalyzed aryl amidation, Xu [/bib_ref] [bib_ref] Synthesis of quinazolin-4(3H)-ones via Pd(II)-catalyzed intramolecular C(sp2)-H carboxamidation of N-arylamidines, Ma [/bib_ref] [bib_ref] Synthetic applications of Pd(II)-catalyzed C-H carboxylation and mechanistic insights: Expedient routes to..., Giri [/bib_ref]. Malacria, and co-workers devised an ingenious radical cascade for making quinazolinones starting from N-acyl-N-(2-iodobenzyl)cyanamides 88 [bib_ref] Radical cyclization of N-acylcyanamides: Total synthesis of luotonin A, Servais [/bib_ref] [bib_ref] Radical migration of substituents of aryl groups on quinazolinones derived from N-acyl..., Larraufie [/bib_ref] [bib_ref] Unprecedented aromatic homolytic substitutions and cyclization of amide-iminyl radicals: Experimental and theoretical..., Beaume [/bib_ref]. Iminyl radicals 89 were obtained by 5-exo-cyclisation onto the nitrile group of the first-formed aryl radicals. Subsequent ring closure took place efficiently onto a variety of aromatic and heteroaromatic rings yielding quinazolin-2-ones 90 (Scheme 16). The method was subsequently extended to the analogous 2-azidoalkyl-N-acylcyanamides 91 and these yielded guanidines 92 [bib_ref] Radical synthesis of guanidines from N-acyl cyanamides, Larraufie [/bib_ref]. The disadvantages of this Scheme 15. Microwave-assisted preparations of quinazoline derivatives from (2-aminoaryl)alkanone O-phenyl oximes [bib_ref] 2-(Aminoaryl)alkanone O-phenyl oximes: Versatile reagents for syntheses of quinazolines, Portela-Cubillo [/bib_ref] [bib_ref] Microwave-promoted syntheses of quinazolines and dihydroquinazolines from 2-aminoarylalkanone O-phenyl oximes, Portela-Cubillo [/bib_ref].
Inclusion of ZnCl 2 , a known promoter of imine formation [bib_ref] First synthesis of marine alkaloid (˘)-bengacarboline, Pouilhes [/bib_ref] , in practice led directly to the corresponding fully aromatic quinazolines 87. Good to excellent yields were obtained with aliphatic aldehydes, aromatic aldehydes having either electron-withdrawing or electron releasing substituents and with heterocyclic aldehydes. The method was also tolerant of substituents in the aminoaryl rings. It's likely that, in addition to encouraging imine formation, the ZnCl 2 forms a complex with the iminyl N-atom in radical cation 86. The acidity of the H-atom adjacent to the cation would thereby be increased, thus facilitating proton-coupled electron transfer (PCET) and thence to the quinazolines 87 (Scheme 15).
Conventional organic preparations of quinazolinones usually start with derivatives of anthranilic acid [bib_ref] Synthesis of quinazolinones and quinazolines, Connolly [/bib_ref] [bib_ref] Synthesis of 2,3-disubstituted-quinazolin-4-(3H)-ones, Patil [/bib_ref]. Several good Pd-and Cu-catalyzed methods for compounds containing this structure have also been published [bib_ref] Synthesis of 3-substituted and 2,3-disubstituted quinazolinones via Cu-catalyzed aryl amidation, Xu [/bib_ref] [bib_ref] Synthesis of quinazolin-4(3H)-ones via Pd(II)-catalyzed intramolecular C(sp2)-H carboxamidation of N-arylamidines, Ma [/bib_ref] [bib_ref] Synthetic applications of Pd(II)-catalyzed C-H carboxylation and mechanistic insights: Expedient routes to..., Giri [/bib_ref]. Malacria, and co-workers devised an ingenious radical cascade for making quinazolinones starting from N-acyl-N-(2-iodobenzyl)cyanamides 88 [bib_ref] Radical cyclization of N-acylcyanamides: Total synthesis of luotonin A, Servais [/bib_ref] [bib_ref] Radical migration of substituents of aryl groups on quinazolinones derived from N-acyl..., Larraufie [/bib_ref] [bib_ref] Unprecedented aromatic homolytic substitutions and cyclization of amide-iminyl radicals: Experimental and theoretical..., Beaume [/bib_ref]. Iminyl radicals 89 were obtained by 5-exo-cyclisation onto the nitrile group of the first-formed aryl radicals. Subsequent ring closure took place efficiently onto a variety of aromatic and heteroaromatic rings yielding quinazolin-2-ones 90 (Scheme 16). The method was subsequently extended to the analogous 2-azidoalkyl-N-acylcyanamides 91 and these yielded guanidines 92 [bib_ref] Radical synthesis of guanidines from N-acyl cyanamides, Larraufie [/bib_ref]. The disadvantages of this methodology were the need to use toxic cyanogen bromide in the syntheses of the precursors and the stoichiometric amounts of organotin hydride needed for the ring closures. Aware that 5-aryl-4,5-dihydro-1,2,4-oxadiazoles 93 are readily available, either from 1,3-dipolar cycloadditions of nitrile oxides to aldimines, or from reactions of amidoximes with carbonyls [bib_ref] Orbital control in the 1,3-dipolar cycloaddition of benzonitrile oxide to benzylideneanilines, Alcaide [/bib_ref] [bib_ref] Synthesis, spectroscopic studies and the mechanism of formation of 4,5-dihydro-1,2,4-oxadiazoles, Srivastava [/bib_ref] , Chiba and co-workers adopted these as their iminyl radical precursors [bib_ref] Oxidative radical skeletal rearrangement induced by molecular oxygen: Synthesis of quinazolinones, Wang [/bib_ref]. They discovered that on treatment in DMSO solution simply with dry air at 120 °C, oxidative rearrangements of 93 were set in motion and resulted in formation of quinazolinones 96 (Scheme 17). Iminyl radicals 94 were generated either via a SET and proton loss process, or by abstraction of the tertiary benzylic H-atom followed by β-scission (Scheme 17). The dominant ring closure was in the 6-endo mode yielding cyclohexadienyl radicals 95 that were oxidised to the quinazolinone products 96 usually in very good yields. The process was tolerant of electron withdrawing and electron releasing substituents, as well Scheme 16. Cascade preparations of quinazolinones and guanidines [bib_ref] Radical cyclization of N-acylcyanamides: Total synthesis of luotonin A, Servais [/bib_ref] [bib_ref] Radical migration of substituents of aryl groups on quinazolinones derived from N-acyl..., Larraufie [/bib_ref] [bib_ref] Unprecedented aromatic homolytic substitutions and cyclization of amide-iminyl radicals: Experimental and theoretical..., Beaume [/bib_ref] [bib_ref] Radical synthesis of guanidines from N-acyl cyanamides, Larraufie [/bib_ref].
Aware that 5-aryl-4,5-dihydro-1,2,4-oxadiazoles 93 are readily available, either from 1,3-dipolar cycloadditions of nitrile oxides to aldimines, or from reactions of amidoximes with carbonyls [bib_ref] Orbital control in the 1,3-dipolar cycloaddition of benzonitrile oxide to benzylideneanilines, Alcaide [/bib_ref] [bib_ref] Synthesis, spectroscopic studies and the mechanism of formation of 4,5-dihydro-1,2,4-oxadiazoles, Srivastava [/bib_ref] , Chiba and co-workers adopted these as their iminyl radical precursors [bib_ref] Oxidative radical skeletal rearrangement induced by molecular oxygen: Synthesis of quinazolinones, Wang [/bib_ref]. They discovered that on treatment in DMSO solution simply with dry air at 120˝C, oxidative rearrangements of 93 were set in motion and resulted in formation of quinazolinones 96 (Scheme 17). Iminyl radicals 94 were generated either via a SET and proton loss process, or by abstraction of the tertiary benzylic H-atom followed by β-scission (Scheme 17). The dominant ring closure was in the 6-endo mode yielding cyclohexadienyl radicals 95 that were oxidised to the quinazolinone products 96 usually in very good yields. The process was tolerant of electron withdrawing and electron releasing substituents, as well as 5-heteroaryl substituents. Substrates with orthoor meta-substituents R 3 gave rise to mixtures of isomeric quinazolinones (see structure 99).
## Scheme 16.
Cascade preparations of quinazolinones and guanidines [bib_ref] Radical cyclization of N-acylcyanamides: Total synthesis of luotonin A, Servais [/bib_ref] [bib_ref] Radical migration of substituents of aryl groups on quinazolinones derived from N-acyl..., Larraufie [/bib_ref] [bib_ref] Unprecedented aromatic homolytic substitutions and cyclization of amide-iminyl radicals: Experimental and theoretical..., Beaume [/bib_ref] [bib_ref] Radical synthesis of guanidines from N-acyl cyanamides, Larraufie [/bib_ref].
Aware that 5-aryl-4,5-dihydro-1,2,4-oxadiazoles 93 are readily available, either from 1,3-dipolar cycloadditions of nitrile oxides to aldimines, or from reactions of amidoximes with carbonyls [bib_ref] Orbital control in the 1,3-dipolar cycloaddition of benzonitrile oxide to benzylideneanilines, Alcaide [/bib_ref] [bib_ref] Synthesis, spectroscopic studies and the mechanism of formation of 4,5-dihydro-1,2,4-oxadiazoles, Srivastava [/bib_ref] , Chiba and co-workers adopted these as their iminyl radical precursors [bib_ref] Oxidative radical skeletal rearrangement induced by molecular oxygen: Synthesis of quinazolinones, Wang [/bib_ref]. They discovered that on treatment in DMSO solution simply with dry air at 120 °C, oxidative rearrangements of 93 were set in motion and resulted in formation of quinazolinones 96 (Scheme 17). Iminyl radicals 94 were generated either via a SET and proton loss process, or by abstraction of the tertiary benzylic H-atom followed by β-scission (Scheme 17). The dominant ring closure was in the 6-endo mode yielding cyclohexadienyl radicals 95 that were oxidised to the quinazolinone products 96 usually in very good yields. The process was tolerant of electron withdrawing and electron releasing substituents, as well as 5-heteroaryl substituents. Substrates with ortho-or meta-substituents R 3 gave rise to mixtures of isomeric quinazolinones (see structure 99). Scheme 17. Preparations of quinazolinones from 4,5-dihydro-1,2,4-oxadiazoles [bib_ref] Oxidative radical skeletal rearrangement induced by molecular oxygen: Synthesis of quinazolinones, Wang [/bib_ref].
Research with precursor 4,5-dihydro-1,2,4-oxadiazoles with ortho-R 3 , or containing 5-pyridine units, indicated that spiro-cyclisation to radicals 97 could compete. In this case an alternative ring opening to carbamoyl radicals 98 also took place and these species ring closed either to quinazolinone isomers 99 or in 5-exo-mode onto a 3-phenyl substituent with production of 3-(arylimino)isoindolin-1-one by-products 100.
## Preparations of quinoxaline derivatives
The quinoxaline and quinoxalin-2-one structural units are also associated with many natural products and biologically active substances [bib_ref] Piperazine imidazo[1,5-a]quinoxaline ureas as high-affinity GABAA ligands of dual functionality, Jacobsen [/bib_ref] [bib_ref] Structure-activity studies of substituted quinoxalinones as multiple-drug-resistance antagonists, Lawrence [/bib_ref] [bib_ref] Synthesis and biological activity of quinoxaline derivatives, Padvi [/bib_ref] [bib_ref] Drug likeness and selective functionalization of quinoxalines, Ferreira [/bib_ref]. These rings have been assembled in several ways based on initial addition of C-, or S-centered radicals to aromatic isonitriles 101. Nanni and coworkers generated cyano-substituted alkyl radicals by treatment of 4-iodobutanenitrile 102 with Scheme 17. Preparations of quinazolinones from 4,5-dihydro-1,2,4-oxadiazoles [bib_ref] Oxidative radical skeletal rearrangement induced by molecular oxygen: Synthesis of quinazolinones, Wang [/bib_ref].
Research with precursor 4,5-dihydro-1,2,4-oxadiazoles with ortho-R 3 , or containing 5-pyridine units, indicated that spiro-cyclisation to radicals 97 could compete. In this case an alternative ring opening to carbamoyl radicals 98 also took place and these species ring closed either to quinazolinone isomers 99 or in 5-exo-mode onto a 3-phenyl substituent with production of 3-(arylimino)isoindolin-1-one by-products 100.
## Preparations of quinoxaline derivatives
The quinoxaline and quinoxalin-2-one structural units are also associated with many natural products and biologically active substances [bib_ref] Piperazine imidazo[1,5-a]quinoxaline ureas as high-affinity GABAA ligands of dual functionality, Jacobsen [/bib_ref] [bib_ref] Structure-activity studies of substituted quinoxalinones as multiple-drug-resistance antagonists, Lawrence [/bib_ref] [bib_ref] Synthesis and biological activity of quinoxaline derivatives, Padvi [/bib_ref] [bib_ref] Drug likeness and selective functionalization of quinoxalines, Ferreira [/bib_ref]. These rings have been assembled in several ways based on initial addition of C-, or S-centered radicals to aromatic isonitriles 101. Nanni and co-workers generated cyano-substituted alkyl radicals by treatment of 4-iodobutanenitrile 102 with hexamethylditin in t-butylbenzene at 150˝C [bib_ref] Radical annulations with nitriles: Novel cascade reactions of cyano-substituted alkyl and sulfanyl..., Camaggi [/bib_ref]. Additions to the isonitriles generated imidoyl radicals 103 that ring closed onto the pendant cyano group in 5-exo mode to afford cyclic iminyl radicals 104. The cascade continued with a further cyclisation on to the aromatic nucleus followed by oxidative re-aromatization to produce cyclopentaquinoxalines 105 in moderate yields (Scheme 18). Significantly, the 2-cyanophenyl disulfide 106 was employed in an analogous process that was free of organotin compounds. UV photolysis of 106, together with an isonitrile, in benzene solution at r.t. gave rise to benzo [bib_ref] Experimental and computational approaches to estimate solubility and permeability in drug discovery..., Lipinski [/bib_ref] [bib_ref] The art and practice of structure-based drug design: A molecular modelling perspective, Bohacek [/bib_ref] thieno quinoxalines 108 in fair yields via α-thioimidoyl radicals and polycyclic-iminyl radicals 107 (Scheme 18).
More recently, photoredox catalyzed versions of the isonitrile insertion process requiring only visible light, have been deployed for quinoxaline preparations. Sun et al. demonstrated that diethyl 2-bromo-2-(2-isocyanoethyl)malonate 109 was a suitable precursor for 3-cyano-alkyl radicals when irradiated by blue LED light in the presence of fac-Ir(ppy) 3 as PC. Cyclopenta-iminyl radicals were obtained as intermediates and afforded cyclopentaquinoxalines 110 in moderate to excellent yields [bib_ref] Synthesis of fused quinoline and quinoxaline derivatives enabled by domino radical triple..., Sun [/bib_ref] (Scheme 18). Jamison and co-workers generated alkyl radicals from readily accessible phenyliodine(III) dicarboxylates [bib_ref] Synthetic uses of organohypervalent iodine compounds through radical pathways, Togo [/bib_ref] with visible light and fac-Ir(ppy) 3 in DMF [bib_ref] Synthesis of highly functionalized polycyclic quinoxaline derivatives using visible-light photoredox catalysis, He [/bib_ref]. Their substrates were aromatic isonitriles with ortho-pyrrole or analogous heteroarene substituents 111. The alkyl radicals coupled with the isonitriles giving imidoyl radical intermediates 112 that ring closed to pyrrolo[1,2-a]quinoxalines (and related structures) 113 without the intermediacy of iminyl radicals (Scheme 18). The process was tolerant of many substituent types enabling a good range of polycyclic heteroarenes to be prepared. Efficiency was enhanced by their development of a continuous flow system that integrated isonitrile formation with the photo-redox cascade. visible light, have been deployed for quinoxaline preparations. Sun et al. demonstrated that diethyl 2-bromo-2-(2-isocyanoethyl)malonate 109 was a suitable precursor for 3-cyano-alkyl radicals when irradiated by blue LED light in the presence of fac-Ir(ppy)3 as PC. Cyclopenta-iminyl radicals were obtained as intermediates and afforded cyclopentaquinoxalines 110 in moderate to excellent yields [bib_ref] Synthesis of fused quinoline and quinoxaline derivatives enabled by domino radical triple..., Sun [/bib_ref] (Scheme 18). Jamison and co-workers generated alkyl radicals from readily accessible phenyliodine(III) dicarboxylates [bib_ref] Synthetic uses of organohypervalent iodine compounds through radical pathways, Togo [/bib_ref] with visible light and fac-Ir(ppy)3 in DMF [bib_ref] Synthesis of highly functionalized polycyclic quinoxaline derivatives using visible-light photoredox catalysis, He [/bib_ref]. Their substrates were aromatic isonitriles with ortho-pyrrole or analogous heteroarene substituents 111. The alkyl radicals coupled with the isonitriles giving imidoyl radical intermediates 112 that ring closed to pyrrolo[1,2-a]quinoxalines (and related structures) 113 without the intermediacy of iminyl radicals (Scheme 18). The process was tolerant of many substituent types enabling a good range of polycyclic heteroarenes to be prepared. Efficiency was enhanced by their development of a continuous flow system that integrated isonitrile formation with the photo-redox cascade. [bib_ref] Radical annulations with nitriles: Novel cascade reactions of cyano-substituted alkyl and sulfanyl..., Camaggi [/bib_ref] [bib_ref] Synthesis of fused quinoline and quinoxaline derivatives enabled by domino radical triple..., Sun [/bib_ref] [bib_ref] Synthesis of highly functionalized polycyclic quinoxaline derivatives using visible-light photoredox catalysis, He [/bib_ref].
## Scheme 18. preparations of quinoxalines via aromatic isonitrile insertion routes
## Iminyl radical mediated preparations of natural products and bioactive compounds
A significant number of iminyl radical based preparative methods have been developed for one or more stages in total or partial syntheses of natural products and bioactive compounds. Trispheridine 114 occurs naturally in flowering plants of the family Amaryllidaceae [bib_ref] Alkaloids from Narcissus asturiensis, Viladomat [/bib_ref] [bib_ref] New crinine-type alkaloids with inhibitory effect on induction of inducible nitric oxide..., Abdel-Halim [/bib_ref]. Trispheridine itself, as well as related compounds, are known to possess anti-tumor activity, high anti-retroviral activity [bib_ref] Alkaloids from leucojum vernum and antiretroviral activity of amaryllidaceae alkaloids, Szlavik [/bib_ref] and neuro-protective activity [bib_ref] Neuroprotective activity and acetylcholinesterase inhibition of five amaryllidaceae species: A comparative study, Cortes [/bib_ref]. At least six iminyl radical-mediated preparations of trispheridine have been devised and these are summarized in Scheme 19.
## Iminyl radical mediated preparations of natural products and bioactive compounds
A significant number of iminyl radical based preparative methods have been developed for one or more stages in total or partial syntheses of natural products and bioactive compounds. Trispheridine 114 occurs naturally in flowering plants of the family Amaryllidaceae [bib_ref] Alkaloids from Narcissus asturiensis, Viladomat [/bib_ref] [bib_ref] New crinine-type alkaloids with inhibitory effect on induction of inducible nitric oxide..., Abdel-Halim [/bib_ref]. Trispheridine itself, as well as related compounds, are known to possess anti-tumor activity, high anti-retroviral activity [bib_ref] Alkaloids from leucojum vernum and antiretroviral activity of amaryllidaceae alkaloids, Szlavik [/bib_ref] and neuro-protective activity [bib_ref] Neuroprotective activity and acetylcholinesterase inhibition of five amaryllidaceae species: A comparative study, Cortes [/bib_ref]. At least six iminyl radical-mediated preparations of trispheridine have been devised and these are summarized in Scheme 19.
Three of these rely on UVA irradiations; all of easily prepared precursors including dioxime oxalate 115 [bib_ref] Dioxime oxalates; new iminyl radical precursors for syntheses of N-heterocycles, Portela-Cubillo [/bib_ref] , O-acyl oxime ester 116 [bib_ref] Photochemistry of acyloximes: Synthesis of heterocycles and natural products, Alonso [/bib_ref] and O-phenyl oxime carbonate 117 [bib_ref] UV promoted phenanthridine syntheses from oxime carbonate derived iminyl radicals, Mcburney [/bib_ref]. All three precursors were made in high yields, essentially in three steps, from commercially available 6-bromopiperonal. The highest reported yield (59%) was that from dioxime oxalate 115 but it is probable that yields from all three processes could be increased by optimization studies. The µwave promoted process from O-phenyl oxime ester 118 was short, clean and comparatively efficient (70%) [bib_ref] Microwave-assisted syntheses of N-heterocycles using alkenone-, alkynone-and aryl-carbonyl O-phenyl oximes: Formal synthesis..., Portela-Cubillo [/bib_ref]. Compound 118 was made in high yield in two steps from 6-bromopiperonal and the reaction conditions were clean and straightforward. The two photoredox catalyzed methods starting from aldehyde 119 [bib_ref] Visible-light-promoted and one-pot synthesis of phenanthridines and quinolines from aldehydes and O-acyl..., An [/bib_ref] and trifluoromethylbenzoyl oxime ester 120 [bib_ref] Visible-light-promoted iminyl-radical formation from acyl oximes: A unified approach to pyridines, quinolines,..., Jiang [/bib_ref] had the highest reported yields and reaction conditions were benign (Scheme 19). The fac-Ir(ppy) 3 catalyzed preparation, starting from aldehyde 119, took only two steps from 6-bromopiperonal. It should be noted, however, that the co-reactant O-(4-cyanobenzoyl)hydroxylamine is not commercial; although it can be made in two high-yielding steps from 4-cyanobenzoyl chloride. and straightforward. The two photoredox catalyzed methods starting from aldehyde 119 [bib_ref] Visible-light-promoted and one-pot synthesis of phenanthridines and quinolines from aldehydes and O-acyl..., An [/bib_ref] and trifluoromethylbenzoyl oxime ester 120 [bib_ref] Visible-light-promoted iminyl-radical formation from acyl oximes: A unified approach to pyridines, quinolines,..., Jiang [/bib_ref] had the highest reported yields and reaction conditions were benign (Scheme 19). The fac-Ir(ppy)3 catalyzed preparation, starting from aldehyde 119, took only two steps from 6-bromopiperonal. It should be noted, however, that the co-reactant O-(4-cyanobenzoyl)hydroxylamine is not commercial; although it can be made in two high-yielding steps from 4-cyanobenzoyl chloride. Vasconine 123, along with assoanine, oxoassoanine, pratosine and ismine, constitute another group of alkaloids, isolated from Narcissus plants of the Amaryllidaceae family, that contain phenanthridine rings [bib_ref] New syntheses of the Amaryllidaceae alkaloids vasconine, assoanine, oxoassoanine, pratosine and ismine..., Rosa [/bib_ref] [bib_ref] Concise synthesis of narcissus pyrrolophenanthridine alkaloids: Vasconine, Assoanine and Oxoassoanine, Parnes [/bib_ref]. The acyl oxime 121 was targeted as a key compound and prepared in six steps from commercial 2-(3-bromophenyl)ethanol [bib_ref] Photochemistry of acyloximes: Synthesis of heterocycles and natural products, Alonso [/bib_ref]. UV irradiation of this through Pyrex in MeCN solution afforded phenanthridinylethanol derivative 122 in 53% yield (Scheme 20). Compound 122 can be converted to vasconine 123 by treatment with PBr3 and can also act as precursor for assoanine, oxoassoanine, and pratosine [bib_ref] New syntheses of the Amaryllidaceae alkaloids vasconine, assoanine, oxoassoanine, pratosine and ismine..., Rosa [/bib_ref].
Noravicine 126a and nornitidine 126b are bioactive benzo[c]phenanthridine alkaloids with antitumor activity [bib_ref] Synthesis of derivatives of NK109, 7-OH benzo[c]phenanthridine alkaloid, and evaluation of their..., Nakanishi [/bib_ref] [bib_ref] Revision of the structure of Fagaridine based on comparison of UV and..., Nakanishi [/bib_ref]. Zhang, Yu and co-workers devised short (4-steps from methylenedioxynaphthalene 124) and high yielding total syntheses employing their photoredox strategy with oxime esters 125a and b [bib_ref] Visible-light-promoted iminyl-radical formation from acyl oximes: A unified approach to pyridines, quinolines,..., Jiang [/bib_ref] (Scheme 20). Scheme 19. Iminyl radical mediated preparations of trispheridine.
Vasconine 123, along with assoanine, oxoassoanine, pratosine and ismine, constitute another group of alkaloids, isolated from Narcissus plants of the Amaryllidaceae family, that contain phenanthridine rings [bib_ref] New syntheses of the Amaryllidaceae alkaloids vasconine, assoanine, oxoassoanine, pratosine and ismine..., Rosa [/bib_ref] [bib_ref] Concise synthesis of narcissus pyrrolophenanthridine alkaloids: Vasconine, Assoanine and Oxoassoanine, Parnes [/bib_ref]. The acyl oxime 121 was targeted as a key compound and prepared in six steps from commercial 2-(3-bromophenyl)ethanol [bib_ref] Photochemistry of acyloximes: Synthesis of heterocycles and natural products, Alonso [/bib_ref]. UV irradiation of this through Pyrex in MeCN solution afforded phenanthridinylethanol derivative 122 in 53% yield (Scheme 20). Compound 122 can be converted to vasconine 123 by treatment with PBr 3 and can also act as precursor for assoanine, oxoassoanine, and pratosine [bib_ref] New syntheses of the Amaryllidaceae alkaloids vasconine, assoanine, oxoassoanine, pratosine and ismine..., Rosa [/bib_ref]. Scheme 20. Iminyl radical strategies for vasconine [bib_ref] Photochemistry of acyloximes: Synthesis of heterocycles and natural products, Alonso [/bib_ref] , noravicine and nornitidine [bib_ref] Visible-light-promoted iminyl-radical formation from acyl oximes: A unified approach to pyridines, quinolines,..., Jiang [/bib_ref].
Natural products that contain the indolizino[1,2-b]quinolin-9(11H)-one structure 130a include camptothecine, mappicine and the nothapodytines. Camptothecine has been obtained from the bark of Camptotheca acuminata (Camptotheca, Happy tree, Tupelo family) [bib_ref] Antifungal activity of camptothecin, trifolin, and hyperoside isolated from camptotheca acuminata, Li [/bib_ref] and mappicine from Mappia foetida miers (Olacaceae family) [bib_ref] Mappicine, a minor alkaloid from Mappia foetida miers, Tuticorin [/bib_ref]. The high bioactivity of these compounds stimulated the production of many synthetic and semi-synthetic analogues [bib_ref] Design and synthesis of new 7-(N-substituted-methyl)-camptothecin derivatives as potent cytotoxic agents, Zhao [/bib_ref] [bib_ref] Synthesis, cytotoxic activity, DNA topoisomerase-II inhibition, molecular modeling and structure-activity relationship of..., Loza-Mejia [/bib_ref]. Several pharmaceuticals containing this core have been licensed for use including irinotecan (Camptosar ® , Campto ® ) which is used for the treatment of colon cancer and Topotecan (trade name Hycamtin ® ) which is approved for treatment of several cancers including ovarian and lung types [bib_ref] Design, synthesis and development of novel camptothecin drugs, Liew [/bib_ref]. A number of total syntheses, incorporating important radical-mediated steps, have been reported. Of particular note are those of Curran and co-workers, most of which have key steps involving amidoyl radicals obtained from additions to aromatic isonitriles [bib_ref] Asymmetric total synthesis of (20R)-homocamptothecin, substituted homocamptothecins and homosilatecans, Gabarda [/bib_ref] [bib_ref] The novel silatecan 7-tert-butyldimethylsilyl-10-hydroxycamptothecin displays high lipophilicity, improved human blood stability, and..., Dom [/bib_ref] [bib_ref] Silylcamptothecins (silatecans): A new family of camptothecin antitumor agents, Josien [/bib_ref] [bib_ref] Tandem radical reactions of isonitriles with 2-pyridonyl and other aryl radicals: Scope..., Curran [/bib_ref] [bib_ref] Cascade radical reactions of isonitriles: A second-generation synthesis of (20S)-camptothecin, topotecan, irinotecan,..., Curran [/bib_ref] [bib_ref] New 4 + 1 radical annulations. A formal total synthesis of (˘)-camptothecin, Liu [/bib_ref] [bib_ref] Solution-phase preparation of a 560-compound library of individual pure mappicine analogues by..., Zhang [/bib_ref]. Scheme 20. Iminyl radical strategies for vasconine [bib_ref] Photochemistry of acyloximes: Synthesis of heterocycles and natural products, Alonso [/bib_ref] , noravicine and nornitidine [bib_ref] Visible-light-promoted iminyl-radical formation from acyl oximes: A unified approach to pyridines, quinolines,..., Jiang [/bib_ref].
Noravicine 126a and nornitidine 126b are bioactive benzo[c]phenanthridine alkaloids with antitumor activity [bib_ref] Synthesis of derivatives of NK109, 7-OH benzo[c]phenanthridine alkaloid, and evaluation of their..., Nakanishi [/bib_ref] [bib_ref] Revision of the structure of Fagaridine based on comparison of UV and..., Nakanishi [/bib_ref]. Zhang, Yu and co-workers devised short (4-steps from methylenedioxynaphthalene 124) and high yielding total syntheses employing their photoredox strategy with oxime esters 125a and b [bib_ref] Visible-light-promoted iminyl-radical formation from acyl oximes: A unified approach to pyridines, quinolines,..., Jiang [/bib_ref] (Scheme 20).
Natural products that contain the indolizino[1,2-b]quinolin-9(11H)-one structure 130a include camptothecine, mappicine and the nothapodytines. Camptothecine has been obtained from the bark of Camptotheca acuminata (Camptotheca, Happy tree, Tupelo family) [bib_ref] Antifungal activity of camptothecin, trifolin, and hyperoside isolated from camptotheca acuminata, Li [/bib_ref] and mappicine from Mappia foetida miers (Olacaceae family) [bib_ref] Mappicine, a minor alkaloid from Mappia foetida miers, Tuticorin [/bib_ref]. The high bioactivity of these compounds stimulated the production of many synthetic and semi-synthetic analogues [bib_ref] Design and synthesis of new 7-(N-substituted-methyl)-camptothecin derivatives as potent cytotoxic agents, Zhao [/bib_ref] [bib_ref] Synthesis, cytotoxic activity, DNA topoisomerase-II inhibition, molecular modeling and structure-activity relationship of..., Loza-Mejia [/bib_ref]. Several pharmaceuticals containing this core have been licensed for use including irinotecan (Camptosar ® , Campto ® ) which is used for the treatment of colon cancer and Topotecan (trade name Hycamtin ® ) which is approved for treatment of several cancers including ovarian and lung types [bib_ref] Design, synthesis and development of novel camptothecin drugs, Liew [/bib_ref]. A number of total syntheses, incorporating important radical-mediated steps, have been reported. Of particular note are those of Curran and co-workers, most of which have key steps involving amidoyl radicals obtained from additions to aromatic isonitriles [bib_ref] Asymmetric total synthesis of (20R)-homocamptothecin, substituted homocamptothecins and homosilatecans, Gabarda [/bib_ref] [bib_ref] The novel silatecan 7-tert-butyldimethylsilyl-10-hydroxycamptothecin displays high lipophilicity, improved human blood stability, and..., Dom [/bib_ref] [bib_ref] Silylcamptothecins (silatecans): A new family of camptothecin antitumor agents, Josien [/bib_ref] [bib_ref] Tandem radical reactions of isonitriles with 2-pyridonyl and other aryl radicals: Scope..., Curran [/bib_ref] [bib_ref] Cascade radical reactions of isonitriles: A second-generation synthesis of (20S)-camptothecin, topotecan, irinotecan,..., Curran [/bib_ref] [bib_ref] New 4 + 1 radical annulations. A formal total synthesis of (˘)-camptothecin, Liu [/bib_ref] [bib_ref] Solution-phase preparation of a 560-compound library of individual pure mappicine analogues by..., Zhang [/bib_ref].
Bowman and co-workers implemented an iminyl radical cascade synthetic protocol for the tetracyclic rings A-D of heterocycles containing the indolizino[1,2-b]quinolin-9(11H)-one 130a unit [bib_ref] Cascade radical synthesis of heteroarenes via iminyl radicals, Bowman [/bib_ref] [bib_ref] Synthesis of heteroarenes using cascade radical cyclisation via iminyl radicals, Bowman [/bib_ref] (Scheme 21). The starting vinyl iodides 127 were assembled in four steps from cinnamaldehyde derivatives and cyanopyridones and then UV irradiated with hexamethylditin in t-BuPh at 150˝C. The initial vinyl radicals 128 ring closed onto the nitrile group in 5-exo mode producing iminyl radicals 129 that underwent cyclisation and re-aromatization to afford tetracycles 130a-c. Scheme 20. Iminyl radical strategies for vasconine [bib_ref] Photochemistry of acyloximes: Synthesis of heterocycles and natural products, Alonso [/bib_ref] , noravicine and nornitidine [bib_ref] Visible-light-promoted iminyl-radical formation from acyl oximes: A unified approach to pyridines, quinolines,..., Jiang [/bib_ref].
Natural products that contain the indolizino[1,2-b]quinolin-9(11H)-one structure 130a include camptothecine, mappicine and the nothapodytines. Camptothecine has been obtained from the bark of Camptotheca acuminata (Camptotheca, Happy tree, Tupelo family) [bib_ref] Antifungal activity of camptothecin, trifolin, and hyperoside isolated from camptotheca acuminata, Li [/bib_ref] and mappicine from Mappia foetida miers (Olacaceae family) [bib_ref] Mappicine, a minor alkaloid from Mappia foetida miers, Tuticorin [/bib_ref]. The high bioactivity of these compounds stimulated the production of many synthetic and semi-synthetic analogues [bib_ref] Design and synthesis of new 7-(N-substituted-methyl)-camptothecin derivatives as potent cytotoxic agents, Zhao [/bib_ref] [bib_ref] Synthesis, cytotoxic activity, DNA topoisomerase-II inhibition, molecular modeling and structure-activity relationship of..., Loza-Mejia [/bib_ref]. Several pharmaceuticals containing this core have been licensed for use including irinotecan (Camptosar ® , Campto ® ) which is used for the treatment of colon cancer and Topotecan (trade name Hycamtin ® ) which is approved for treatment of several cancers including ovarian and lung types [bib_ref] Design, synthesis and development of novel camptothecin drugs, Liew [/bib_ref]. A number of total syntheses, incorporating important radical-mediated steps, have been reported. Of particular note are those of Curran and co-workers, most of which have key steps involving amidoyl radicals obtained from additions to aromatic isonitriles [bib_ref] Asymmetric total synthesis of (20R)-homocamptothecin, substituted homocamptothecins and homosilatecans, Gabarda [/bib_ref] [bib_ref] The novel silatecan 7-tert-butyldimethylsilyl-10-hydroxycamptothecin displays high lipophilicity, improved human blood stability, and..., Dom [/bib_ref] [bib_ref] Silylcamptothecins (silatecans): A new family of camptothecin antitumor agents, Josien [/bib_ref] [bib_ref] Tandem radical reactions of isonitriles with 2-pyridonyl and other aryl radicals: Scope..., Curran [/bib_ref] [bib_ref] Cascade radical reactions of isonitriles: A second-generation synthesis of (20S)-camptothecin, topotecan, irinotecan,..., Curran [/bib_ref] [bib_ref] New 4 + 1 radical annulations. A formal total synthesis of (˘)-camptothecin, Liu [/bib_ref] [bib_ref] Solution-phase preparation of a 560-compound library of individual pure mappicine analogues by..., Zhang [/bib_ref].
Bowman and co-workers implemented an iminyl radical cascade synthetic protocol for the tetracyclic rings A-D of heterocycles containing the indolizino[1,2-b]quinolin-9(11H)-one 130a unit [bib_ref] Cascade radical synthesis of heteroarenes via iminyl radicals, Bowman [/bib_ref] [bib_ref] Synthesis of heteroarenes using cascade radical cyclisation via iminyl radicals, Bowman [/bib_ref] (Scheme 21). The starting vinyl iodides 127 were assembled in four steps from cinnamaldehyde derivatives and cyanopyridones and then UV irradiated with hexamethylditin in t-BuPh at 150 °C. The initial vinyl radicals 128 ring closed onto the nitrile group in 5-exo mode producing iminyl radicals 129 that underwent cyclisation and re-aromatization to afford tetracycles 130a-c. Scheme 21. Radical-mediated preparations of indolizino[1,2-b]quinolin-9(11H)-ones [bib_ref] Cascade radical synthesis of heteroarenes via iminyl radicals, Bowman [/bib_ref] [bib_ref] Synthesis of heteroarenes using cascade radical cyclisation via iminyl radicals, Bowman [/bib_ref].
Luotonins A, B, E (132a-c) and congeners are structurally related to the mappicines and combine the 2,3-dihydro-1H-pyrrolo[3,4-b]quinoline ring system with the quinazolinone unit. They have been isolated from plant species of the genus Peganum found in China and are well-recognized as remedies in traditional Chinese medicine. They are given for disorders including rheumatism, inflammation, influenza, hepatitis and leukemia and are known antagonists for human DNA topoisomerase I [bib_ref] A naturally occurring human DNA topoisomerase I poison, Cagir [/bib_ref] [bib_ref] Regioselective quinazolinone-directed ortho lithiation of quinazolinoylquinoline: Practical synthesis of naturally occurring human..., Mhaske [/bib_ref]. Structure/activity relationships have been investigated for heterocycles of this class [bib_ref] Synthesis and properties of luotonin A homologues and their aza-analogues, Lee [/bib_ref]. Various non-radical syntheses have been devised [bib_ref] Three-step total synthesis of pyrroloquinazolinoquinoline alkaloid, luotonin A, by intramolecular hetero Diels-Alder..., Toyota [/bib_ref] [bib_ref] Microwave-assisted rapid synthesis of the cytotoxic alkaloid luotonin A, Yadav [/bib_ref] [bib_ref] A new synthesis of the cytotoxic alkaloid Luotonine A, Dallavalle [/bib_ref]. Bowman and co-workers adapted their radical cascade protocol for a comparatively short synthesis of luotonin A 123a [bib_ref] Synthesis of heteroarenes using cascade radical cyclisation via iminyl radicals, Bowman [/bib_ref]. The vinyl iodide radical precursor 131 was made by alkylation of 4-oxo-3,4-dihydroquinazoline-2-carbonitrile with a substituted cinnamyl bromide. On UV irradiation of a solution of 131 and hexamethylditin in t-BuPh, luotonin A 132a was isolated in a very modest yield of 21% (Scheme 22). A slightly improved yield Scheme 21. Radical-mediated preparations of indolizino[1,2-b]quinolin-9(11H)-ones [bib_ref] Cascade radical synthesis of heteroarenes via iminyl radicals, Bowman [/bib_ref] [bib_ref] Synthesis of heteroarenes using cascade radical cyclisation via iminyl radicals, Bowman [/bib_ref].
Luotonins A, B, E (132a-c) and congeners are structurally related to the mappicines and combine the 2,3-dihydro-1H-pyrrolo[3,4-b]quinoline ring system with the quinazolinone unit. They have been isolated from plant species of the genus Peganum found in China and are well-recognized as remedies in traditional Chinese medicine. They are given for disorders including rheumatism, inflammation, influenza, hepatitis and leukemia and are known antagonists for human DNA topoisomerase I [bib_ref] A naturally occurring human DNA topoisomerase I poison, Cagir [/bib_ref] [bib_ref] Regioselective quinazolinone-directed ortho lithiation of quinazolinoylquinoline: Practical synthesis of naturally occurring human..., Mhaske [/bib_ref]. Structure/activity relationships have been investigated for heterocycles of this class [bib_ref] Synthesis and properties of luotonin A homologues and their aza-analogues, Lee [/bib_ref]. Various non-radical syntheses have been devised [bib_ref] Three-step total synthesis of pyrroloquinazolinoquinoline alkaloid, luotonin A, by intramolecular hetero Diels-Alder..., Toyota [/bib_ref] [bib_ref] Microwave-assisted rapid synthesis of the cytotoxic alkaloid luotonin A, Yadav [/bib_ref] [bib_ref] A new synthesis of the cytotoxic alkaloid Luotonine A, Dallavalle [/bib_ref]. Bowman and co-workers adapted their radical cascade protocol for a comparatively short synthesis of luotonin A 123a [bib_ref] Synthesis of heteroarenes using cascade radical cyclisation via iminyl radicals, Bowman [/bib_ref]. The vinyl iodide radical precursor 131 was made by alkylation of 4-oxo-3, 4-dihydroquinazoline-2-carbonitrile with a substituted cinnamyl bromide. On UV irradiation of a solution of 131 and hexamethylditin in t-BuPh, luotonin A 132a was isolated in a very modest yield of 21% (Scheme 22). A slightly improved yield (30%) was obtained when the reaction was carried out in a sealed tube, at 120˝C, with added di-t-butyl peroxide.
Courillon, Malacria and co-workers devised an alternative strategy in which the starting material was the N-acyl-N-(2-iodobenzyl)cyanamide 133 [bib_ref] Radical cyclization of N-acylcyanamides: Total synthesis of luotonin A, Servais [/bib_ref] (Scheme 22). For the luotonin synthesis, 3-(azidomethyl)-2-iodoquinoline was prepared in four steps from quinoline chlorcarbaldehyde and then reduced to the corresponding amine and benzoylated to give 133. UV irradiation of the latter, with hexamethylditin, in refluxing toluene for 6 h, gave a 43% yield of luotonin A 132a.
di-t-butyl peroxide.
Courillon, Malacria and co-workers devised an alternative strategy in which the starting material was the N-acyl-N-(2-iodobenzyl)cyanamide 133 [bib_ref] Radical cyclization of N-acylcyanamides: Total synthesis of luotonin A, Servais [/bib_ref] (Scheme 22). For the luotonin synthesis, 3-(azidomethyl)-2-iodoquinoline was prepared in four steps from quinoline chlorcarbaldehyde and then reduced to the corresponding amine and benzoylated to give 133. UV irradiation of the latter, with hexamethylditin, in refluxing toluene for 6 h, gave a 43% yield of luotonin A 132a. The same research team also discovered an organotin-free protocol employing the same N-acyl-N-(2-iodobenzyl)cyanamide 133 [bib_ref] Unprecedented aromatic homolytic substitutions and cyclization of amide-iminyl radicals: Experimental and theoretical..., Beaume [/bib_ref]. When this was UV photolyzed with pyridine (5 equiv.) in refluxing benzene, in air, luotonin A (132a) was obtained in an improved yield of 54%.
Chiba and co-workers successfully adapted their dihydro-1,2,4-oxadiazole method for preparations of several bio-active compounds. Rutaecarpine (sometimes rutecarpine) is an indoloquinazoline alkaloid that has been isolated from Euodia plants of the Rutaceae family and has non-steroidal anti-inflammatory drug (NSAID) activity [bib_ref] Induction of NAD(P)H: Quinone reductase by rutaecarpine isolated from the fruits of..., Ahn [/bib_ref]. The related 2-methoxy-13-methyl-rutaecarpine 136 has antimalarial activity and has been obtained from Araliopsis tabouensis, an African perennial tree [bib_ref] Indoloquinazoline alkaloids from Araliopsis tabouensis, Christopher [/bib_ref]. In their synthesis, Chiba and co-workers prepared dihydro-1,2,4-oxadiazole 135 in two steps from indolyl imino methylthioether 134 [bib_ref] Oxidative radical skeletal rearrangement induced by molecular oxygen: Synthesis of quinazolinones, Wang [/bib_ref]. Then on heating this as a DMSO solution in air the 2-methoxy-13-methylrutaecarpine 136 was obtained in 62% yield (Scheme 23). Scheme 23. Syntheses of rutaecarpine and ispinesib derivatives via dihydro-1,2,4-oxadiazoles [bib_ref] Oxidative radical skeletal rearrangement induced by molecular oxygen: Synthesis of quinazolinones, Wang [/bib_ref].
Ispinesib 140 is a synthetic quinazolinone that is under study as a potential anti-cancer agent [bib_ref] Novel benzimidazole inhibitors bind to a unique site in the kinesin spindle..., Sheth [/bib_ref] [bib_ref] AZD4877), a kinesin spindle protein inhibitor and potential anticancer agent, Theoclitou [/bib_ref]. A similar strategy was employed in a short iminyl radical-mediated synthesis [bib_ref] Oxidative radical skeletal rearrangement induced by molecular oxygen: Synthesis of quinazolinones, Wang [/bib_ref]. Alkylated phthalimide 137 was prepared from L-valine and thence to dihydro-1,2,4-oxadiazole 138 as a mixture of diastereoisomers in three steps. On heating one stereoisomer in aerated DMSO the quinazoline derivative 139, which is a key precursor for ispinesib, was obtained in moderate yield The same research team also discovered an organotin-free protocol employing the same N-acyl-N-(2-iodobenzyl)cyanamide 133 [bib_ref] Unprecedented aromatic homolytic substitutions and cyclization of amide-iminyl radicals: Experimental and theoretical..., Beaume [/bib_ref]. When this was UV photolyzed with pyridine (5 equiv.) in refluxing benzene, in air, luotonin A (132a) was obtained in an improved yield of 54%.
Chiba and co-workers successfully adapted their dihydro-1,2,4-oxadiazole method for preparations of several bio-active compounds. Rutaecarpine (sometimes rutecarpine) is an indoloquinazoline alkaloid that has been isolated from Euodia plants of the Rutaceae family and has non-steroidal anti-inflammatory drug (NSAID) activity [bib_ref] Induction of NAD(P)H: Quinone reductase by rutaecarpine isolated from the fruits of..., Ahn [/bib_ref]. The related 2-methoxy-13-methyl-rutaecarpine 136 has antimalarial activity and has been obtained from Araliopsis tabouensis, an African perennial tree [bib_ref] Indoloquinazoline alkaloids from Araliopsis tabouensis, Christopher [/bib_ref]. In their synthesis, Chiba and co-workers prepared dihydro-1,2,4-oxadiazole 135 in two steps from indolyl imino methylthioether 134 [bib_ref] Oxidative radical skeletal rearrangement induced by molecular oxygen: Synthesis of quinazolinones, Wang [/bib_ref]. Then on heating this as a DMSO solution in air the 2-methoxy-13-methylrutaecarpine 136 was obtained in 62% yield (Scheme 23).
(30%) was obtained when the reaction was carried out in a sealed tube, at 120 °C, with added di-t-butyl peroxide.
Courillon, Malacria and co-workers devised an alternative strategy in which the starting material was the N-acyl-N-(2-iodobenzyl)cyanamide 133 [bib_ref] Radical cyclization of N-acylcyanamides: Total synthesis of luotonin A, Servais [/bib_ref] (Scheme 22). For the luotonin synthesis, 3-(azidomethyl)-2-iodoquinoline was prepared in four steps from quinoline chlorcarbaldehyde and then reduced to the corresponding amine and benzoylated to give 133. UV irradiation of the latter, with hexamethylditin, in refluxing toluene for 6 h, gave a 43% yield of luotonin A 132a. . Iminyl radical mediated syntheses of luotonin A [bib_ref] Radical cyclization of N-acylcyanamides: Total synthesis of luotonin A, Servais [/bib_ref] [bib_ref] Unprecedented aromatic homolytic substitutions and cyclization of amide-iminyl radicals: Experimental and theoretical..., Beaume [/bib_ref] [bib_ref] Synthesis of heteroarenes using cascade radical cyclisation via iminyl radicals, Bowman [/bib_ref].
The same research team also discovered an organotin-free protocol employing the same N-acyl-N-(2-iodobenzyl)cyanamide 133 [bib_ref] Unprecedented aromatic homolytic substitutions and cyclization of amide-iminyl radicals: Experimental and theoretical..., Beaume [/bib_ref]. When this was UV photolyzed with pyridine (5 equiv.) in refluxing benzene, in air, luotonin A (132a) was obtained in an improved yield of 54%.
Chiba and co-workers successfully adapted their dihydro-1,2,4-oxadiazole method for preparations of several bio-active compounds. Rutaecarpine (sometimes rutecarpine) is an indoloquinazoline alkaloid that has been isolated from Euodia plants of the Rutaceae family and has non-steroidal anti-inflammatory drug (NSAID) activity [bib_ref] Induction of NAD(P)H: Quinone reductase by rutaecarpine isolated from the fruits of..., Ahn [/bib_ref]. The related 2-methoxy-13-methyl-rutaecarpine 136 has antimalarial activity and has been obtained from Araliopsis tabouensis, an African perennial tree [bib_ref] Indoloquinazoline alkaloids from Araliopsis tabouensis, Christopher [/bib_ref]. In their synthesis, Chiba and co-workers prepared dihydro-1,2,4-oxadiazole 135 in two steps from indolyl imino methylthioether 134 [bib_ref] Oxidative radical skeletal rearrangement induced by molecular oxygen: Synthesis of quinazolinones, Wang [/bib_ref]. Then on heating this as a DMSO solution in air the 2-methoxy-13-methylrutaecarpine 136 was obtained in 62% yield (Scheme 23). Scheme 23. Syntheses of rutaecarpine and ispinesib derivatives via dihydro-1,2,4-oxadiazoles [bib_ref] Oxidative radical skeletal rearrangement induced by molecular oxygen: Synthesis of quinazolinones, Wang [/bib_ref].
Ispinesib 140 is a synthetic quinazolinone that is under study as a potential anti-cancer agent [bib_ref] Novel benzimidazole inhibitors bind to a unique site in the kinesin spindle..., Sheth [/bib_ref] [bib_ref] AZD4877), a kinesin spindle protein inhibitor and potential anticancer agent, Theoclitou [/bib_ref]. A similar strategy was employed in a short iminyl radical-mediated synthesis [bib_ref] Oxidative radical skeletal rearrangement induced by molecular oxygen: Synthesis of quinazolinones, Wang [/bib_ref]. Alkylated phthalimide 137 was prepared from L-valine and thence to dihydro-1,2,4-oxadiazole 138 as a mixture of diastereoisomers in three steps. On heating one stereoisomer in aerated DMSO the quinazoline derivative 139, which is a key precursor for ispinesib, was obtained in moderate yield Scheme 23. Syntheses of rutaecarpine and ispinesib derivatives via dihydro-1,2,4-oxadiazoles [bib_ref] Oxidative radical skeletal rearrangement induced by molecular oxygen: Synthesis of quinazolinones, Wang [/bib_ref].
Ispinesib 140 is a synthetic quinazolinone that is under study as a potential anti-cancer agent [bib_ref] Novel benzimidazole inhibitors bind to a unique site in the kinesin spindle..., Sheth [/bib_ref] [bib_ref] AZD4877), a kinesin spindle protein inhibitor and potential anticancer agent, Theoclitou [/bib_ref]. A similar strategy was employed in a short iminyl radical-mediated synthesis [bib_ref] Oxidative radical skeletal rearrangement induced by molecular oxygen: Synthesis of quinazolinones, Wang [/bib_ref]. Alkylated phthalimide 137 was prepared from L-valine and thence to dihydro-1,2,4-oxadiazole 138 as a mixture of diastereoisomers in three steps. On heating one stereoisomer in aerated DMSO the quinazoline derivative 139, which is a key precursor for ispinesib, was obtained in moderate yield though with some racemization. These dihydro-1,2,4-oxadiazole rearrangements provide a benign, metal-free and atom-economical protocol with great promise as a general synthetic tool for the incorporation of quinazolinone units.
# Conclusions
Five-and six-membered ring aza-arenes are hugely important in nutrition, medicine and pharmacology because of their many useful therapeutic properties. Short, mild and atom-efficient synthetic methods for compounds containing these units are clearly provided by iminyl radical mediated systems. Although iminyl radicals can be accessed by a variety of ways, carbonyl oximes and oxime ethers are particularly effective non-toxic precursors with long shelf lives. These starting materials lend themselves to 'clean' preparative protocols, free of harsh acid/base or redox reagents, and employing photolysis, µwave irradiation or photoredox catalysis. Iminyl radicals generated by these means and suitably substituted with acceptor units facilitate syntheses of a large variety of such compounds. Suitable acceptor groups include aromatics, heteroarenes, alkenes and alkynes. Reproducible and efficient syntheses of good scope and impressive generality have thereby been devised for diverse families of heterocycles. Significantly, iminyl radical based steps have been chosen for natural products and bioactive molecules containing phenanthridine, indole and quinazoline core units. It seems certain that further variations of these methods will be devised and find additional roles and applications. It can be reliably concluded that iminyl-radical based synthetic methods form a most valuable toolkit to expedite the exploration of 'drug-like' chemical space.
[fig] Scheme 1: Generation of iminyl radicals from oxime derivatives and subsequent ring closures. [/fig]
[fig] Scheme 3: Preparation of dioxime oxalates and their use in dihydropyrrole syntheses[54]. [/fig]
[fig] Scheme 4: Preparation of oxime ethers and microwave-promoted syntheses of dihydropyrroles and pyrroles[56][57][58]. [/fig]
[fig] Scheme 6: Preparation of dihydropyrroles from N-sulfonylimines[61]. [/fig]
[fig] Scheme 7: Iminyl radical 6-endo ring closures onto aromatic acceptors. [/fig]
[fig] Molecules 2016 ,: 21, 660 12 of 23 methodology were the need to use toxic cyanogen bromide in the syntheses of the precursors and the stoichiometric amounts of organotin hydride needed for the ring closures.Scheme 16. Cascade preparations of quinazolinones and guanidines[105][106][107][108]. [/fig]
[fig] Scheme 22: Iminyl radical mediated syntheses of luotonin A[105,107,143]. [/fig]
[table] .: Similarly, biphenyl-2-carbaldehyde O-phenyl oximes 60a and 2-arylnicotinaldehyde O-phenyl oximes 60b, with either electron releasing or electron withdrawing substituents, yielded the corresponding phenanthridine 61a or benzo[h][1,6]naphthyridine derivatives 61b respectively The scope of the method was easily extended such that benzo[k]phenanthridine 63 and benzo[b]thieno [2,3-c]quinoline 65 were prepared in a very few steps and in acceptable yields (Scheme 12) 11 Preparation of phenanthridines from oxime carbamates[78] [/table]
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Cytosolic lipid droplets as engineered organelles for production and accumulation of terpenoid biomaterials in leaves
Supplementary Figure 1. Diterpenoids were sequestered in isolated lipid droplet fractions.Lipid droplet fractions were isolated from N. benthamiana leaves producing either plastid:AgABS alone or in combination with AtWRI1 1-397 and NoLDSP without or with YFP-tag. The different engineering approaches are indicated in a and b (, was included; -, was not included). The top view of the floating lipid droplet layers after gradient centrifugation is shown (a). The diterpenoid content in the isolated lipid droplet fractions is given with open circles representing individual biological replicates and bars representing average values and SD (N=3) (b). Data were analyzed by Shapiro-Wilk, Brown-Forsythe ANOVA (P 0.0302) and Welch ANOVA (P 0.0041) followed by t-tests (unpaired, two-tailed, Welch correction, P<0.05). Statistically significant differences are indicated by a-c based on t-tests. NoLDSP promotes clustering of small lipid droplets. The localization of yellow fluorescent fusion protein-tagged NoLDSP (YFP-NoLDSP) in clustered lipid droplets was imaged by confocal laser scanning microscopy on a collected lipid droplet fraction (c, phase contrast; d, YFP channel, scale bar 50 m; e, YFP channel, scale bar 5 m). Source Data are provided as a Source Data file.
## Supplementary figure 2.
NoLDSP promotes clustering of small lipid droplets. Representative confocal laser scanning microscopy images are shown for N. benthamiana leaves producing either AtWRI1 1-397 (Control) or AtWRI1 1-397 +NoLDSP. Nile red staining and confocal laser scanning microscopy were used to detect lipid droplets in mesophyll cells. Under the experimental conditions, the control accumulated a very low level of triacylglycerol in lipid droplets that was often too low to be visualized by Nile Red staining. Expression of AtWRI1 1-397 +NoLDSP enhanced triacylglycerol accumulation and resulted in clustering of small lipid droplets (green) as indicated by arrows (scale bars: 5 m).
[formula] Name Sequence 5' 3' Description 4b-BamHI-AtWRI1-F GAATGGATCCGGACAATGAAGAAGC insertion of AtWRI1 1-397 into pENTR4 4b-XhoI-AtWRI1-R GAATCTCGAGTCACTCTCTTCCCAC insertion of AtWRI1 1-397 into pENTR4 No-LDSP_F ATGGCCGGCCCCATCATG amplification of NoLDSP No-LDSP+Stop_R TTACTCCTTGAGGAGGAACTTCGCCAC amplification of NoLDSP 6b-NcoI-NoLDSP_F2 CAGTCACCATGGCCGGCCCCATCATG insertion of NoLDSP into pENTR4 6b-EcoRV- NoLDSP+Stop_R2 CCACAGGATATCTTACTCCTTGAGGAGGAACTT CGC insertion of NoLDSP into [/formula]
[fig] Figure 3: LC/MS analysis of a leaf extract. The extract was prepared from a N. benthamiana leaf producing AtWRI1 1-397 +NoLDSP+ElHMGR 159-582 +cytosol:MtGGDPS+LD:AgABS 85-868 +ER:PsCYP720B4. Extracted ion chromatograms m/z 301.217 are shown in acquisition function 1 (0 V) and function 2 (20-80 V). Compounds 1-4 were subjected to MS/MS analysis. The elution order and MS/MS data were consistent with compound 1-3 and compound 4 being formate adducts of tetrahexosyl diterpenoid acid isomers and trihexosyl diterpenoid acid, respectively (Supplementary Figures 4 and 5). Supplementary Figure 4. LC/MS/MS analysis of tetrahexosyl diterpenoid acid isomers (1-3). An extract from a N. benthamiana leaf producing AtWRI1 1-397 +NoLDSP+ElHMGR 159-582 +cytosol:MtGGDPS+LD:AgABS 85-868 +ER:PsCYP720B4 was subjected to LC/MS/MS analysis. Accurate masses and MS/MS spectra of compounds 1-3 are consistent with formate adducts of tetrahexosyl diterpenoid acid isomers [M+formate]m/z 995.4 (fragments: [M-formate]m/z 949.4, [M-formatepartial loss of dihexosyl]m/z 667.3 and [M-formate-tetrahexosyl]m/z 301.2). Supplementary Figure 5. LC/MS/MS analysis of a trihexosyl diterpenoid acid (4). An extract from a N. benthamiana leaf producing AtWRI1 1-397 +NoLDSP+ElHMGR 159-582 +cytosol:MtGGDPS+LD:AgABS 85-868 +ER:PsCYP720B4 was subjected to LC/MS/MS analysis. Elemental composition and MS/MS spectrum of compound 4 are consistent with a formate adduct of trihexosyl diterpenoid acid [M+formate]m/z 833.3 (fragments: [M-formate]m/z 787.4, [M-formate-dihexosyl]m/z 463.3 and [M-formatetrihexosyl]m/z 301.2). [/fig]
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Initial Medication in Patients of Newly Diagnosed Parkinson’s Disease in Taiwan
Introduction: Several treatment guidelines for Parkinson's disease (PD) had been proposed in recent decades. The aim of current study was to investigate the initial medication utilized in newly diagnosed PD subjects in Taiwan during an elevenyear period.Methods: A total of 7,550 patients with newly diagnosed Parkinsonism were retrospectively enrolled from the National Health Insurance Research Database of Taiwan from 2000 to 2010. After excluding patients at risk of secondary or atypical Parkinsonism, those never receiving medication or having incomplete data, 1,645 subjects were included. The participants were then divided into four treating regimen groups, namely levodopa (LD) only group, dopamine agonist (DA) only group, LD+DA group, and No-LD, No-DA group. The demographic data and medication retention rate were compared across the four treatment groups.Results: LD only and No-LD, No-DA regimens were the main initial choice of PD treatment in Taiwan. LD containing drugs were more often prescribed to the elderly population than the other two treatment regimens, while No-LD, No-DA medication was the major initial choice for younger patients. DA only regimen occupied only 3-4% of the initial PD prescriptions and was given predominantly by neurologists. Over the eleven-year period, there is a trend for the middleaged population to receive medication containing LD as initial treatment. The one year retention rate of anti-Parkinsonism medication was around 30-50% in our population. Age, polypharmacy, change of one-year daily levodopa equivalent dosage and newly onset of dementia, stroke and psychiatric diseases all affect drug compliance in PD patients.Conclusions: This is the first long-term study to explore initial pharmacotherapies in an Asian PD population. We hope to provide evidence for adjusting government policies and public education of physicians and PD patients in the future.
# Introduction
Parkinson's disease (PD) is the second most common neurodegenerative disease with the cardinal motor features of bradykinesia, resting tremor, rigidity and postural instability [bib_ref] Accuracy of clinical diagnosis of idiopathic Parkinson's disease: a clinico-pathological study of..., Hughes [/bib_ref]. The incidence and prevalence of PD increase with age. In the United States, the estimated annual incidence and prevalence of PD are 12.3 per 100,000 and 106.9 per 100,000 [bib_ref] Incidence of Parkinson's disease: variation by age, gender, and race/ ethnicity, Van Den Eeden [/bib_ref] [bib_ref] The frequency of idiopathic Parkinson's disease by age, ethnic group, and sex..., Mayeux [/bib_ref].
Current therapies for PD patients include levodopa (LD), dopamine agonist (DA), monoamine oxidase B inhibitor (MAO-B inhibitor), catechol-O-methyltransferase inhibitor (COMT inhibitor), anticholinergic drugs and amantadine [bib_ref] Clinical inquiry. Which drugs work best for early Parkinson's disease?, Jain [/bib_ref] [bib_ref] Therapies in Parkinson's disease, Jankovic [/bib_ref] [bib_ref] The Movement Disorder Society Evidence-Based Medicine Review Update: Treatments for the motor..., Fox [/bib_ref]. After 4-6 years of treatment with LD, 40% of the patients developed motor fluctuations and another 40% had dyskinesia [bib_ref] Frequency of levodopa-related dyskinesias and motor fluctuations as estimated from the cumulative..., Ahlskog [/bib_ref]. The incidence of motor complications was even higher in the younger PD population receiving LD treatment [bib_ref] Frequency of levodopa-related dyskinesias and motor fluctuations as estimated from the cumulative..., Ahlskog [/bib_ref] [bib_ref] Early development of levodopa-induced dyskinesias and response fluctuations in young-onset Parkinson's disease, Kostic [/bib_ref]. In one series, more than 90% of young PD patients receiving therapies containing LD developed motor complications within 5 years [bib_ref] Young onset Parkinson's disease, Quinn [/bib_ref]. As compared to LD, DAs had a lower risk of motor complications but more neuropsychiatric and systemic adverse effects. Therefore, the usage of DAs as an initial therapeutic choice was limited in the elderly patients [bib_ref] Slower progression of Parkinson's disease with ropinirole versus levodopa: The REAL-PET study, Whone [/bib_ref] [bib_ref] Pramipexole vs levodopa as initial treatment for Parkinson disease: a 4-year randomized..., Holloway [/bib_ref] [bib_ref] Levodopa in the treatment of Parkinson's disease: an old drug still going..., Poewe [/bib_ref]. There were many guidelines proposed for the management of PD over the past two decades [bib_ref] Therapies in Parkinson's disease, Jankovic [/bib_ref] [bib_ref] The Movement Disorder Society Evidence-Based Medicine Review Update: Treatments for the motor..., Fox [/bib_ref] [bib_ref] Updates in the medical management of Parkinson disease, Fernandez [/bib_ref] [bib_ref] Initial therapy for Parkinson's disease: levodopa vs. dopamine receptor agonists, Kondo [/bib_ref] [bib_ref] Treatment of early Parkinson's disease, Koller [/bib_ref] [bib_ref] Initiating therapy for Parkinson's disease, Silver [/bib_ref] [bib_ref] The treatment of early Parkinson's disease: levodopa rehabilitated, Vlaar [/bib_ref] [bib_ref] Diagnosis and initiation of treatment in Parkinson's disease, Lyons [/bib_ref]. Several of them suggested using a MAO-B inhibitor in patients with mild disability, DAs in younger patients with moderate to severe disability and LD in elderly patients with prominent functional impairment [bib_ref] Therapies in Parkinson's disease, Jankovic [/bib_ref] [bib_ref] Treatment of early Parkinson's disease, Koller [/bib_ref] [bib_ref] Initiating therapy for Parkinson's disease, Silver [/bib_ref] [bib_ref] Diagnosis and initiation of treatment in Parkinson's disease, Lyons [/bib_ref]. The annual total net ingredient cost for anti-Parkinsonism medication increased gradually from £37 million in 1998 to £130 million in 2010 in England since the introduction of non-ergot DAs and MAO-B inhibitors [bib_ref] Prescribing in Parkinson's disease: a story of hope and adverse events, Morrish [/bib_ref]. However, there have only been a limited number of studies discussing the trend of initial therapeutic choice for PD patients in the real world [bib_ref] Initial pharmacotherapy in a population of veterans with Parkinson disease, Swarztrauber [/bib_ref] [bib_ref] Patterns of initial pharmacotherapy for Parkinson's disease in the United States, Huse [/bib_ref].
The National Health Insurance (NHI) program was implemented in Taiwan since March 1995. It offers a comprehensive, unified and universal health insurance program to all citizens [bib_ref] Trends in the use of antiepileptic drugs in Taiwan from 2003 to..., Hsieh [/bib_ref]. The aim of current study is to explore the initial choice of pharmacotherapies in newly diagnosed PD patients from 2000 to 2010 by using the National Health Insurance Research Database (NHIRD).
# Methods
## Data source
Data was obtained from Taiwan's NHIRD, which is maintained by the National Health Research Institutes (NHRI) and overseen by the state-run Bureau of NHI (BNHI) for research purposes. The NHI program in Taiwan started in 1995 and covered over 99.5% of the population by the end of 2009 [bib_ref] Utilization of statins and aspirin among patients with diabetes and hyperlipidemia, Lin [/bib_ref].
All research had been approved by Institutional Review Board of Taichung Veterans General Hospital (CE13262). No informed consents from participants were obtained because the data were analyzed anonymously. We used data from 2000 to 2010 for the current study. According to the NHIRD, random sampling of the cohort is achieved by using the linear congruential random number generation function of the Sun WorkShop Compiler C 5.0. The distributions of the random samples were representative of the Taiwanese population [bib_ref] Trends in the use of antiepileptic drugs in Taiwan from 2003 to..., Hsieh [/bib_ref].
## Study population
Patients were defined as having Parkinsonism if they received an ICD-9-CM code of 332.x more than three times in the outpatient services or more than once during hospitalization between January 2000 and December 2010. Patients were excluded if (1) they were exposed to medication with a high risk of drug-induced Parkinsonism for more than three months before PD diagnosis [fig_ref] Table 1: Demographic characteristics of newly diagnosed PD patients, 2000-2010 [/fig_ref] in File S1) [bib_ref] Drug-induced parkinsonism in the elderly: incidence, management and prevention, Lopez-Sendon [/bib_ref] , (2) they had diseases potentially causing secondary or atypical Parkinsonism [fig_ref] Table 2: Initial medication choice in patients with newly diagnosed Parkinson's disease according to... [/fig_ref] in File S1) [bib_ref] Differential diagnosis of parkinsonian syndromes: dynamics of time courses are essential, Lachenmayer [/bib_ref] , (3) they had never received any anti-Parkinsonism medication, or [bib_ref] Clinical inquiry. Which drugs work best for early Parkinson's disease?, Jain [/bib_ref] if their data was incomplete. The remaining patients were included in the present study.
## Initial pharmacotherapy
The study participants were divided into four different treatment groups according to the initial medication used within the first year of PD diagnosis. The definitions of the four treatment groups were: (1) LD only group -Patients that received LD only or LD plus other anti-Parkinsonism medication other than DAs; (2) DA only group -Patients who took DAs only or DAs plus other anti-Parkinsonism drugs other than LD; (3) LD+DA group -Subjects who received a combination of LD and DAs regardless with or without other anti-Parkinsonism drugs; and (4) No-LD, No-DA group -Individuals who were given MAO-B inhibitors, anticholinergic drugs or amantadine as initial treatments [fig_ref] Figure 1: Flow chart of selection of PD patients [/fig_ref]. Levodopa containing medication includes LD only and LD+DA drugs. All the anti-Parkinsonism drugs available in Taiwan are listed in [fig_ref] Table 3: Initial medication choice in patients with newly diagnosed Parkinson's disease according to... [/fig_ref] in File S1.
## Variables investigated
Data including age, gender, medical institution, year and doctors' specialty of initial prescriptions, interval between PD diagnosis and prescription, initial and one-year daily levodopa equivalent dosage (LDED), initial drug cost and one year retention rate of anti-Parkinsonism medication were analyzed. Factors affecting the drug compliance of PD patients were also explored.
One-year retention rate was defined as patients received medications confined to each treatment group for more than 6 months divided by the total number of patients in the same An Eleven-Year Retrospective Cohort Study from the NHIRD PLOS ONE | www.plosone.org medication group. Any subject taking extra medication belonging to another treatment regimen group for more than three times was defined as a non-retention patient. For example, a subject in the LD only group was considered to be ''non-retention patient'' if DAs were prescribed for more than 3 times in subsequent followups.
The definition of medication possession ratio (MPR) was the number of days with medication supply divided by 365 days (i.e. the follow-up period in this study). Having an MPR of more than 80% was classified as good compliance [bib_ref] Drug therapies for Parkinson's disease: A database analysis of patient compliance and..., Tarrants [/bib_ref].
# Statistical analysis
Data was expressed as mean 6 standard deviation or number (%). Categorical variables were compared by chi-squared test and continuous variables were compared by one-way analysis of variance (ANOVA). For analyses with significant p value in ANOVA, Scheffe's test was performed for post-hoc analysis to evaluate the difference between groups. For categorical variables, chi-squared test was used for pairwise comparison between either two of the four treatment groups. All analyses were performed using SAS version 9.2 (SAS Institute, Cary, NC, USA). A p value of ,0.05 was considered to be statistically significant.
# Results
A total of 7,550 subjects were diagnosed as having Parkinsonism in the NHIRD, and 1,645 patients fulfilled the inclusion criteria of newly diagnosed PD in the present study. They were assigned into four different regimen groups, including 821 patients in LD only group, 65 persons in DA only group, 58 persons in LD+DA group and 701 persons in No-LD, No-DA group [fig_ref] Figure 1: Flow chart of selection of PD patients [/fig_ref]. The demographic data of the participants is shown in [fig_ref] Table 1: Demographic characteristics of newly diagnosed PD patients, 2000-2010 [/fig_ref]. [fig_ref] Table 1: Demographic characteristics of newly diagnosed PD patients, 2000-2010 [/fig_ref]. The trend was more obvious when the proportion of No-LD, No-DA patients were compared across different age groups (!40, 41-64, and "65 y/o). For subjects younger than 40 y/o, up to 90.6% of PD patients received No-LD, No-DA drugs as initial treatment [fig_ref] Table 2: Initial medication choice in patients with newly diagnosed Parkinson's disease according to... [/fig_ref]. For the other two age groups, the percentage of No-LD, No-DA users dropped to 49.9% in middle age group and 30.9% in elderly age group respectively (all p values ,0.0001).
## Age as a major determinant of initial drug choice
## Medical institutions and doctors' specialty affected initial prescription
Most patients (51.4%) received their first anti-PD medication in regional hospitals [fig_ref] Table 1: Demographic characteristics of newly diagnosed PD patients, 2000-2010 [/fig_ref]. Physicians in medical centers and regional hospitals had similar treating philosophy for PD (p = 0.219, df = 2). Majority of the DA only regimen was prescribed by doctors in the medical centers (43.1%) and in regional hospitals (50.7%), while only 6.2% of DA only regimen was prescribed by doctors in the clinics. 48.1% of PD patients [bib_ref] Initial pharmacotherapy in a population of veterans with Parkinson disease, Swarztrauber [/bib_ref].7% for DA+LD group respectively). The initial drugs choices for newly diagnosed PD were similar between neurologist in medical centers and those in regional hospitals (p = 0.805, df = 6, [fig_ref] Table 3: Initial medication choice in patients with newly diagnosed Parkinson's disease according to... [/fig_ref]. [fig_ref] Figure 1: Flow chart of selection of PD patients [/fig_ref].
[formula] N (%) N (%) N (%) N (%) N (%)#40 [/formula]
## Time trend of initial prescription and interval between pd diagnosis and medical intervention
Most PD medication (81.3%) was prescribed immediately after the patients were diagnosed with PD [fig_ref] Table 1: Demographic characteristics of newly diagnosed PD patients, 2000-2010 [/fig_ref]. Among the four treatment groups, LD only regimen was more frequently chosen (51.3%). For subjects receiving DA only regimen, up to one-third (32.3%) had more than one month delay between the PD diagnosis and initiation of medical therapy [fig_ref] Table 1: Demographic characteristics of newly diagnosed PD patients, 2000-2010 [/fig_ref]. [fig_ref] Table 1: Demographic characteristics of newly diagnosed PD patients, 2000-2010 [/fig_ref] Similar trend was found in the one-year daily LDED analysis. (p,0.05 in the post-hoc analysis, [fig_ref] Table 1: Demographic characteristics of newly diagnosed PD patients, 2000-2010 [/fig_ref].
## Drug cost and dosage
## Drug compliance by one-year retention rate
The one year retention rate was around 30-50% in four treatment groups and there were no statistical differences among them (p = 0.07, [fig_ref] Table 1: Demographic characteristics of newly diagnosed PD patients, 2000-2010 [/fig_ref]. We then analyzed the retention rate of each treatment group after six to twelve months of initial treatment [fig_ref] Table 4: Medication of non-retention patients between 6 and 12 months after starting treatment [/fig_ref]. The most common reason for non-retention was medication withdrawal, and it accounted for 66.5% of the non-retention patients. 244 patients (25.9%) in the non-retention group needed medication containing LD (LD only or LD+DA medication) during the follow-up period (25.0% in LD only group, 34.1% in DA only group, 24.1% in LD+DA group and 26.2% in No-LD, No-DA group). No patient in the DA only group shifted back to a DA only regimen once non-retained.
Determinants of one-year retention rate -age, doctors' specialty, medical institutions, one-year drug dosage and neuropsychiatric comorbidities Patients younger than 65 years-old had a significantly higher retention rate as compared to those older than 65 years-old (46.7% and 40.3% respectively, p,0.05, [fig_ref] Table 5: Clinical characteristics of retention and non-retention groups of patients with newly diagnosed... [/fig_ref].
Using patients with MPR$80% as a reference, more PD patients who received initial treatment from neurologists and medical centers would shift to other drugs during the one year follow-up period (p,0.05). The ratio of shifting medication was 23.0% in medical centers, 18.6% in regional hospitals and 19.1% in clinics. 27.3% of patients receiving initial medication from neurologists would shift medication during the study period while 11.6% of subjects with initial drugs from non-neurologists would change their drugs. Patients taking medication from multiple medical institutions had the highest ratio of no medication after one year of starting treatment in comparison to those receiving major treatment ($50% of medication) from the same level of medical institutions (82.9% in multiple hospitals, 46.2% in clinics, 37.1% in regional hospitals and 28.6% in medical centers, p, 0.05).
Pot-hoc analysis showed no significant differences of initial daily LDED in retention and non-retention patients. Patients in the groups of MPR,80% and shift-medication had a higher one-year daily LDED in comparison to subject with MPR$80% and those without medication (318.76356. [bib_ref] Early development of levodopa-induced dyskinesias and response fluctuations in young-onset Parkinson's disease, Kostic [/bib_ref] Mortality and most systemic morbidities, such as CNS trauma, sepsis, congestive heart failure, liver decompensation, renal failure and neoplasms, didn't affect drug compliance of PD patients. However, patients in the shift-medication group had a significantly higher percentage of newly onset of stroke (37.1%), dementia (22.9%) and psychiatric diseases (50.8%) when compared to those with MPR$80%.
# Discussion
This is the first long-term study to explore the initial pharmacotherapies in an Asian PD population. The differences with regards to treatment strategies for newly diagnosed PD patients were compared between Taiwan PD patients and the proposed guidelines for Western populations. Factors affecting initial drug choice and medical compliance were analyzed.
The initial drug choices preference for newly diagnosed young PD patients was different between Taiwan and Western countries. Doctors in Taiwan preferred to give No-LD, No-DA medication for younger patients (60.6%), and the ratio was significantly higher than the percentage in Western populations (7-33%) [bib_ref] Initial pharmacotherapy in a population of veterans with Parkinson disease, Swarztrauber [/bib_ref] [bib_ref] Patterns of initial pharmacotherapy for Parkinson's disease in the United States, Huse [/bib_ref]. About 15-20% of young PD patients (,65 y/o) in Western countries used DAs as their initial treatment and the percentage was much higher than that in the present study (4.2%). There are a number of possible explanations for this phenomenon. First, the BNHI has strict budget limitations for doctors which could predispose physicians to choose more cost-effective medications. Second, seeking medical service was more convenient and cheaper for Taiwanese patients when compared to patients in the United States. Patients would be more likely to change their doctors if their initial medication was less potent or caused more adverse effects. Third, the demographic features of study participants were different between current study and the related literature reports. The percentage of patients younger than 65 years-old (39.5%) was higher in the present research as compared to the studies of Kari Swarztrauber et al. and Daniel M. Huse et al. [bib_ref] Initial pharmacotherapy in a population of veterans with Parkinson disease, Swarztrauber [/bib_ref] [bib_ref] Patterns of initial pharmacotherapy for Parkinson's disease in the United States, Huse [/bib_ref]. The ratio of participants treated by neurologists in our study was more than 1.5 times when compared to the study by Kari Swarztrauber et al. Patterns of initial pharmacotherapies in PD patients changed in Taiwan during the passing eleven years. The major difference included decreasing usage of No-LD, No-DA regimen and increasing LD usage as initial PD regimen in patients of 41-64 years-old. Patients of this age group are the major economic support of their family. As a result, they might need more effective drugs for symptomatic control. To balance the patient needs and budget limitations, LD containing regimen is the preferred regimen for most doctors in Taiwan.
Compared to Western PD populations, the one year retention rate was lower (30-50%) in Taiwan. However, the determinants of medication compliance were similar between Western PD patients and subjects in Taiwan [bib_ref] Drug therapies for Parkinson's disease: A database analysis of patient compliance and..., Tarrants [/bib_ref] [bib_ref] Medication adherence and associated outcomes in medicare health maintenance organization-enrolled older adults..., Kulkarni [/bib_ref] [bib_ref] Non-completion of changes to prescribed medications in people with Parkinson's disease, Ng [/bib_ref] [bib_ref] Suboptimal medication adherence in Parkinson's disease, Grosset [/bib_ref]. In the related literature, the medication possession ratio in newly treated PD patients was around 50-75% during the first year [bib_ref] Drug therapies for Parkinson's disease: A database analysis of patient compliance and..., Tarrants [/bib_ref] [bib_ref] Medication adherence and associated outcomes in medicare health maintenance organization-enrolled older adults..., Kulkarni [/bib_ref]. In the present study, only 32.5% of patients could maintain medication uninterrupted during their first year [bib_ref] Drug therapies for Parkinson's disease: A database analysis of patient compliance and..., Tarrants [/bib_ref]. Subjects with older ages, initial prescribing year between 2006 and 2010, receiving their first drugs from non-neurologists, receiving drugs from institutions other than medical centers, polypharmacy, higher one-year daily LDED and neuropsychiatric comorbidities such as stroke, dementia and psychiatric disorders had suboptimal medical persistence. Gender, mortality rate and other systemic comorbidities didn't influence medical possession condition of PD population in Taiwan. Participants who received medication from medical centers and neurologists more frequently changed their medication during follow-up. The better availability of different regimens in these medical institutions might be one of the explanations for this finding. 25.9% of non-retention patients changed their medication to a LD containing regimen. The percentage of early PD patients who needed to add LD in LD-naïve therapy within two years was 17.2% in the REAL-PET study and 53% in the CALM-PD study [bib_ref] Slower progression of Parkinson's disease with ropinirole versus levodopa: The REAL-PET study, Whone [/bib_ref] [bib_ref] Pramipexole vs levodopa as initial treatment for Parkinson disease: A randomized controlled..., Study [/bib_ref]. The medication withdraw rate was up to 38.1% among all patients. Although we established a strict exclusion criteria, some patients with secondary or atypical Parkinsonism such as drugrelated Parkinsonism or dementia with Lewy bodies might still be included in our analysis [bib_ref] Drug-induced parkinsonism in the elderly: incidence, management and prevention, Lopez-Sendon [/bib_ref] [bib_ref] Differential diagnosis of parkinsonian syndromes: dynamics of time courses are essential, Lachenmayer [/bib_ref]. Some doctors may use levodopa in the beginning to observe the clinical response for diagnostic purposes. These would also contribute to the low retention rate in this study.
There were a number of limitations that should be highlighted with regards to our studies. First, we could not obtain the medical charts from the NHIRD. Therefore, we could not completely exclude the possibility to include secondary Parkinsonism or atypical Parkinsonism in our study participants. We acknowledge this would affect the results of one-year retention rate because patients with dementia of Lewy bodies have a poorer drug response and higher risk of encountering neuropsychiatric adverse effects. However, doctors might still treat patients who had only Parkinsonism symptoms and didn't show overt cognitive decline as PD patients in the beginning. Second, we were not sure about the functional status of Taiwanese PD patients who started seeking medical help which would influence initial medical choice and subsequent drug titration. We analyzed the medications of these subjects after one year of starting treatment which might reflect the truly clinical status and suitable medication for these patients. Third, we didn't include all participants in NHIRD for analysis. However, a prior study showed that the distributions of the sampling results were representative of the Taiwanese population [bib_ref] Trends in the use of antiepileptic drugs in Taiwan from 2003 to..., Hsieh [/bib_ref].
# Conclusion
This is the first retrospective research to discuss the initial medication in newly diagnosed PD patients in an Asian population and the possible explanations for divergence of treatment of our patients from guidelines proposed by specialists. Although, further reports from other countries are needed to establish a clear picture about PD treatment in the real world, we hope to provide evidence f The medical institution from where patients received $50% medication during the first year. Subjects would be classified into multiple hospitals group if they received medication ,50% from each level of medical institution. g Only patients with medication from outpatient services were analyzed. h The last prescription of patients with medication from outpatient services during one-year follow-up were analyzed. i Definition of comorbidities: patients with diseases of the following ICD codes for more than three times during the outpatient services or once during hospitalization within one year after the diagnosis of Parkinson's disease. to encourage the adjustment of government policies and public education of physicians and PD patients in the future. [fig_ref] Figure 1: Flow chart of selection of PD patients [/fig_ref] Trends of initial pharmacotherapies with doctors' specialty and medical institutions during the eleven years. (Compared by chi-squared test) (A1-2) Neurologists from centers (p = 0.127), (B1-2) Non-neurologists from centers (p = 0.637), (C1-2) Neurologists from regional hospitals (p = 0.319), (D1-2) Non-neurologists from regional hospitals (p = 0.963), (E1-2) Clinics (p = 0.182). (TIF)
## Supporting information
File S1 [fig_ref] Table 1: Demographic characteristics of newly diagnosed PD patients, 2000-2010 [/fig_ref] in File S1. Drugs with high risk of extrapyramidal symptoms. [fig_ref] Table 2: Initial medication choice in patients with newly diagnosed Parkinson's disease according to... [/fig_ref] in File S1. Diseases with risk of of 2nd or atypical Parkinsonism. [fig_ref] Table 3: Initial medication choice in patients with newly diagnosed Parkinson's disease according to... [/fig_ref] in File S1.
Available medication for treating Parkinson's disease in Taiwan between 2000-2010.
(DOCX)
[fig] Figure 1: Flow chart of selection of PD patients. doi:10.1371/journal.pone.0107465.g001 [/fig]
[fig] Figure 2: Trends of initial pharmacotherapies in different age groups between 2000-2005 and 2006-2010. (Compared by chi-squared test) (A1) #40 years-old, 2000-2005, (A2) #40 years-old, 2006-2010 (p = 0.643), (B1) 41-64 years-old, 2000-2005, (B2) 41-64 years-old, 2006-2010 (p = 0.042), (C1) $65 years-old, 2000-2005, (C2) $65 years-old, 2006-2010 (p = 0.099 [/fig]
[table] Table 1: Demographic characteristics of newly diagnosed PD patients, 2000-2010. LD only group differs from No-LD, No-DA group; g DA only group differs from No-LD, No-DA group; h LD+DA group differs from LD only group. *p,0.05 compared to LD only group; # p,0.05 compared to medical center group; @p,0.05 compared to day 0 group. PD: Parkinson's disease, N: case number, %: percentage, SD: standard deviation, NTD: New Taiwan Dollar. doi:10.1371/journal.pone.0107465.t001 [/table]
[table] Table 2: Initial medication choice in patients with newly diagnosed Parkinson's disease according to age, 2000-2010. [/table]
[table] Table 3: Initial medication choice in patients with newly diagnosed Parkinson's disease according to prescribing doctor, 2000-2010. [/table]
[table] Table 4: Medication of non-retention patients between 6 and 12 months after starting treatment. [/table]
[table] Table 5: Clinical characteristics of retention and non-retention groups of patients with newly diagnosed Parkinson's disease. [/table]
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Inventory and Analysis of Controlled Trials of Mobile Phone Applications Targeting Substance Use Disorders: A Systematic Review
Background: Less than 20% of people with addictions have access to adequate treatment. Mobile health could improve access to care. No systematic review evaluates effectiveness of mobile health applications for addiction.Objectives: First aim was to describe controlled trials evaluating the effectiveness of smartphone applications targeting substance use disorders and addictive behaviors. Secondly, we aimed to understand how the application produced changes in behavior and craving management.Method: A systematic review based on PRISMA recommendations was conducted on MEDLINE, CENTRAL, and PsycINFO. Studies had to be controlled trials concerning addictive disorders (substance/behavior), mobile application-based interventions, assessing effectiveness or impact of those applications upon use, published after 2008. Relevant information was systematically screened for synthesis. Quality and risk of bias were evaluated with JADAD score.Results: Search strategy retrieved 22 articles (2014-2019) corresponding to 22 applications targeting tobacco, alcohol, other substances and binge eating disorder. Control groups had access to usual treatments or a placebo-application or no treatment. Eight applications showed reduced use. Most of the applications informed about risks of use and suggestions for monitoring use. Twelve applications managed craving.Discussion: Heterogeneity limited study comparisons. Duration of studies was too short to predict sustainable results. A reduction of craving seemed related to a reduction in use.Conclusion: There is a lack of robust and comparable studies on mHealth applications for addiction treatment. Such applications could become significant contributors in clinical practice in the future so longer-termed double-blind studies are needed. Targeting craving to prevent relapse should be systematic.
# Introduction
Substance use disorder (SUD) and behavioral addiction are a major public health concern affecting 10-15% of the world population. Regarding legal and illegal substance use, worldwide prevalence has remained globally stable over the last few years. Even though European countries have the highest prevalence of alcohol and tobacco consumption, a decrease has been observed over the past decade. However, global health burden remains substantial. Beyond significant costs in health care, SUD human cost is alarming: more than 7 million deaths per year for tobacco, more than 3 million for alcohol and 450,000 for other substances.
Addiction is a chronic disorder which persists beyond abstinence. More than withdrawal symptoms, craving is considered to be a major contributor to repeated relapses. Craving is listed as one of the core diagnostic criteria in the DSM 5, placing it as a main symptom of addiction and a legitimate target for treatment (10). Despite addiction being a severe condition, it is estimated that overall less than 20% of people with an addictive disorder have access to adequate treatment and this is true across countries.
Mobile health (mHealth) may help to reduce this "treatment gap" by improving early diagnosis and access to treatment. It has been defined by the World Health Organization as any medical intervention based on mobile devices. With the dissemination of mobile services in developed and developing countries, patients with chronic diseases are particularly concerned worldwide. These technologies represent a considerable opportunity to access people in need of medical help, where and when it can be difficult in practice. mHealth is a means to overcome social and territorial disparities in health . The digitalization of healthcare services has improved access to information, professional support and medical assistance due to its asynchronous means of communication that abolishes barriers such as traveling time and costs and schedule conflicts with healthcare professionals. mHealth can also contribute to raise awareness among young substance users who consider themselves in good health, while this population is very much affected by risky behaviors. In France, only 12% of teenagers have never experienced tobacco, alcohol or cannabis; revealing a high accessibility of these substancesand this is true also in Europe, North America, and Australia. Smartphones, as an autonomous tool, may be a promising new direction to improve the commitment of young adults to care and improving self-efficacy and empowerment.
In clinical practice, health applications may offer a complementary approach to usual direct contact care. The combined application of cognitive behavioral therapy (CBT) with smartphone applications could increase access to effective interventions. Immediate intervention through these applications can enhance therapeutic effectiveness, consolidate and maintain behavioral change on a long-term basis, thus, helping the patient to be independent without being isolated from professional support.
While more than 300,000 health applications currently exist, including hundreds of "cessation support" applications, few of them have been clinically tested before being put on the market. The majority of these applications have no proof of validity. Some applications even encourage substance use, implicitly or explicitly. Yet, studies suggest that mobile applications can positively influence our health behavior. Some web-based or instant messaging interventions have demonstrated short-to mid-term effectiveness in reducing use. To date, no systematic review has evaluated the effectiveness of mobile applications for the treatment of addictive disorders. The aim of this literature review was to identify and describe controlled treatment trials on mobile health applications which support behavior change among users with problematic behaviors or substance uses by the reduction of use or abstinence. Secondly, we aimed to understand how the application produced changes in use or behavior and the management of craving.
# Methods
## Review protocol
This systematic literature review was based on the ≪ Preferred Reporting Items for Systematic Reviews and Meta-analyses ≫ (PRISMA) recommendations.
## Information source and search
Keywords were defined around 4 criteria (mobile applications, use disorder, effectiveness, excluding web-based interventions) and linked by Boolean operators to generate the following MESH equation: ((smartphone app * OR mobile app * )) AND ((substance use disorder OR addict * OR addictive behavior OR cessation OR recovery OR craving OR alcohol OR smok * OR tobacco OR cannabis OR marijuana OR heroin OR cocaine OR opioid OR gambl * OR binge eating OR porn)) AND ((efficacy OR effectiveness OR impact OR validity)) NOT ((web-based intervention OR Internet)).
## Eligibility criteria and study selection
Studies were included if they met the following criteria: controlled trials concerning addictive disorders (substance/behavior), mobile application-based interventions, assessing the effectiveness or impact of those applications upon use. Addictive behaviors already added in DSM-5 (pathological gambling) or to be considered for further revisions because of important clinical data and research progress [binge eating, pornography] were considered in this review. The research was limited to English and French articles that were published after 2008 which corresponds to the year of first release of health applications.
Age, sex, or nationality of the sample population were not included in the selection criteria. Articles doing a descriptive review of mobile applications, study protocols with no results on efficacy, and literature reviews were excluded.
The articles were first screened by their title and abstract. If relevant, full-text articles were read entirely for a second level selection. Database access and reference management were done by Endnote X6 software.
## Data collection process and synthesis
Each selected paper was screened for relevant information such as whether the application treated one or more addictions, its functionalities, the target population, the randomization, the characteristics of the control group(s) and the results on use and/or craving. The quality of the study and risk of bias was evaluated by the JADAD score (41); a good methodological quality was defined by a score above 3/5.
# Results
## Study selection
The initial search found 1,713 articles. After screening by titles and abstracts, 34 articles were retained and thoroughly reviewed. Twenty-two controlled studies met our eligibility criteria. The selection steps are shown in . The 22 selected articles concerned 22 applicationsfocused on: tobacco (12 articles), alcohol (8 articles) , other substances (1 article), and binge eating disorder (BED) (1 article). The "A-CHESS" and "SmartQuit" (version 1.0 and 2.1) applications were each studied by two different teams . One study evaluated two alcohol cessation applications ("Promillekoll" and "PartyPlanner"). No application was dedicated to multiple addictions.
The majority of studies were published since 2017 (n = 18) and a minority (n = 4) in 2014 and 2015, no studies were published before. The detailed analysis of each study is presented in Tables 3-5. A total of 39,031 participants were included in the studies (tobacco: 34,174; alcohol: 4,716; other substances than alcohol and tobacco: 75; BED: 66) whose duration ranged from 1 to 12 months (average 5 months). The studies were conducted in 11 different countries. Two studies included participants aged 16 and overand the other studies included adults.
## Tobacco addiction applications
## Characteristics of studies
One study targeted users aged 19-29 years (25). One application was specifically intended to support pregnant women (52) and another one for aboriginal Australian population. The level of severity of addiction varied between studies. Two studies included people with high severity (Heaviness smoking index (HSI) > 5, Fagerström> 7)and two others, with medium severity (his = 3, Fagerström = 5).
One application was compared to a self-help guide with similar contents to the application (25), three applications were compared to group therapy or brief interventionand two other control groups did not have access to any intervention. Other studies compared the active application to a placebo version of the application, mainly for informational or monitoring purposes [;].
## Effectiveness of applications
The evaluation criteria of the studies were self-reported abstinenceor biologically verified abstinence (expired /urinary level of carbon monoxide (CO))or self-reported reduction in use.
The rate of abstinence for the "Quit Advisor Plus" application was at 28.5% at 1 month compared to 10.2% at 6-month follow-up. Nevertheless, the overall quit rate for "Quit Advisor Plus" and for the remaining 2,214 participants of "SmokeFree, " who had variable nicotine dependence levels, was significantly higher compared to the placebo application, at 6 and 3 RCT, ITT, 3-arm parallel groups: Combined group (Smartquit + "ACT") (n = 50) v/s "ACT" group (n = 50) v/s BCT group (n = 50). 6 weeks and 90 min sessions of "ACT" and BCT Biochemical verification of abstinence at 6 weeks and 6 months Abstinence: At 6 weeks: 36% combined group v/s 20% "ACT" group v/s 24% BCT group (p > 0.05) At 6 months: 24% combined group v/s 24% "ACT" group v/s 20% BCT group (p > 0.05) Reduction of consumption: Significant decrease for "SmartQuit" group at 6 weeks compared to "ACT" group (p = 0.017) and combined group (p = 0.013). Not significant at 6 months (p = 0.930 and p = 0.759 respectively) Not evaluated
[formula] (Continued) [/formula]
Frontiers in Psychiatry | www.frontiersin.org. The other studies did not specify the reduction of use [;].
## Functionalities of applications
The main functions of the applications were information on the risks of tobacco use, the benefits of abstinence, the different modes of cessation and monitoring of tobacco use, financial savings and health gains due to quitting. Some applications had special features, such as personalization of data, the particular modes of detecting tobacco useor specific craving management techniques [ ;].
## Impact of interventions on craving
Among the eight applications which managed craving, three of themevaluated the effectiveness of the intervention on this symptom. Unlike "PhoS", "SmartQuit 1.0" (43) showed a positive impact on craving with a higher quit rate and "Craving to Quit" (47), a reduction in craving intensity as well as a decrease in the association between craving and tobacco use.
## Methodological quality of studies-risk of bias
Two studies had low methodological quality (JADAD score < 3) (46, 49) due to a non-optimal randomizationor the lack of description of randomization and double-blinding. Five studies were single-blinded [(44-46, 50, 52);].
## Alcohol addiction applications
## Characteristics of studies
Three studies aimed University studentsor users aged 16-25 years (60) engaged in hazardous drinking [Alcohol Use Disorder Test (AUDIT) score >6 for women, >8 for men, >4 drinks per event]. Other studies included users with alcohol use disorder (AUD) according to the DSM IV (55, 56) and 5 (59) criteria or AUDIT score >12. Two studies recruited patients with AUD who were discharged from residential treatment programs . Two applications were compared to the usual CBT program or brief intervention , and one application was compared to a placebo version which was non-customizable and for information purposes only (54). One control group had delayed access, at 1 month, to the application (60) and three others had no intervention [ ;].
## Effectiveness of applications
The main outcome was alcohol reduction in quantity and/or frequency, either self-reported or evaluated by the AUDIT score and/or the daily drinking questionnaire (DDQ). Complete alcohol cessation was sought in three studies .
Three studies, concerning the two applications "A-CHESS" (55, 56) and "LBMI-A" (59) found a significant reduction in use
App: Noom monitor Functionalities: -Self-monitoring: exercise, meal/snacks, compensatory behaviors, craving, weight -Personal notes Specificities: -Combined use with behavioral cognitive therapy (self-help guide) Control group: -Behavioral cognitive therapy only app, application; cig, cigarettes; eBAC, estimated blood alcohol concentration; US, United States (of America).
as well as an increase in abstinence, among patients who were diagnosed with AUD, at 12 months and 6 weeks, respectively. Using "A-CHES" application was also positively correlated with a better adherence to outpatient addiction treatment. A positive correlation between the use of "A-CHESS" and the decrease in risky drinking days was found. The "TeleCoach"application showed a significant decrease in the frequency of use, without any impact on the quantity, at 3-month follow-up while "Ray's Night Out" (60) showed a reduction in the quantity of alcohol use only at 1 month assessment. The "Promillekoll" application that estimated the blood alcohol concentration (BAC), showed a higher frequency of alcohol use. Compared to the control group, no change in drinking behavior were reported in the other studies [;].
## Functionalities of the applications
Most of the applications informed about the consequences of risky drinking and monitored the number of drinks. Three applications estimated the BAC (53, 58). One application used cognitive bias retraining by alcohol eviction games to review the user's approach to alcohol use. Three applications prevented situations of high risk of relapse by identifying craving in real time . The self-determination theory which aimed at developing competency, relatedness, and autonomy was used in one application [(55, 56);].
## Impact of interventions on craving
Although three applications managed craving, the effectiveness on its reduction was not sought in the corresponding studies: "A-CHESS" (55, 56), "LBMI-A", and "TeleCoach" [(57);].
## Methodological quality of studies-risk of bias
Five studies were of good methodological quality (JADAD score > 3) (55-58, 60). One study was not randomized. None of the studies were double-blinded.
## Other substances addiction application
## Characteristics of study
Only one study concerning the "S-Health" application was identified. The participants were users of other substances (ex: heroin, amphetamines...) in methadone treatment for opioid addiction .
The control group had access to a placebo version of the application that only provided information on use by instant messages.
## Effectiveness of application
The number of days of use was self-reported daily and a multidrug urine test was performed weekly by a clinician. A significant decrease in the number of days of use was observed in the interventional group. However, the positivity of the urine test was not statistically different between the two groups.
## Functionalities of the application
Multiple surveys were conducted daily, systematically or upon request, about the context and personal state in which craving occurred, its expression and subject's response. The application also informed about reduction of HIV risk behavior and provided educational materials by text messages.
## Impact of interventions on craving
Even if the application dealt with craving, the impact of the intervention on this symptom was not sought, only substance use was reported.
## Methodological quality of study-risk of bias
This study had a low methodological quality (JADAD score = 1). The method of randomization was incorrect as the distribution of participants between the two groups was uneven. The study was not conducted in double-blind condition.
## Binge eating addiction application
## Characteristics of study
Only one study concerning the "Noom monitor" application was found. Participants were eligible if they met the diagnostic criteria of bulimia nervosa or binge eating disorder according to the DSM 5 or DSM IV with once weekly binge eating and/or purging.
The combined use of the application to behavioral cognitive therapy (BCT) was compared to BCT alone .
## Effectiveness of application
The change in eating disorder behavior, with or without compensatory behaviors was evaluated by the Eating Disorder Examination Questionnaire (EDE-Q). No significant difference was found in the decrease of binge eating episodes or compensatory behaviors in both arms.
## Functionalities of the application
The application served as a self-monitoring tool to record activities (physical exercises, meals/snacks, compensatory behaviors, craving, weight, personal notes;.
## Impact of interventions on craving
The effect of the intervention on craving was not evaluated in this study.
## Methodological quality of study-risk of bias
This double-blind study had a good methodological quality [JADAD score = 3;].
# Discussion
## Synthesis of the main results
The aim of this systematic review of the literature was to identify and describe published controlled trials concerning health applications which support addiction behavior change among problem users, to substance or behavior addictions. We identified 22 trials regarding 22 applications. Each application targeted a unique addiction: tobacco, alcohol, other substances, and binge eating. The results of this review suggest that very few of these applications have shown compelling evidences of their efficacy upon abstinence or reduction of use or craving.
## Critical analysis of effective applications
A total of 8 applications reported results supporting effectiveness (3 for tobacco, 4 for alcohol, and 1 for other substances use). Among the smoking addiction applications, "Quit Advisor Plus", "SmokeFree", and "SmokeBeat" (46) showed significant change in use behavior compared to controls, at 6, 3, and 1 month, respectively. Even if the rate of abstinence was higher for "Quit Advisor Plus, " a constant decline in quit rates was observed throughout the study. The number of abstinent participants was, numerically, half as important at 6-month follow-up which implies that more than half of them relapsed. The two other studies had important attrition bias. The retention rates were extremely low (7.5%) for "SmokeFree, " despite massive recruitment, leading to inequalities between the characteristics of the study arms. Disparities were also found for "SmokeBeat", where the control group had a higher tobacco addiction level due to error in randomization, which could, partially, explain the lack of effectiveness of the intervention in this group. Both "SmokeFree"and "SmokeBeat" (46) were conducted over a too short duration to predict sustainable results on efficacy. Furthermore, no sub-group analysis was made to determine which level of severity of addiction could be more receptive to this type of intervention. For applications which treated AUD, "A-CHESS" was positively correlated with improvement of use behavior among diagnosed patients, during aftercare, over 12 months. Findings in both studies were consistent. A better compliance to treatment was found in the intervention group. "A-CHESS" was the only application whose study was conducted during aftercare which represents a crucial moment for therapeutic adherence . The impact of this application could be explained by the mechanism of behavior change based on self-efficacy and the fact that patients were encouraged to be proactive in their care by seeking social support in critical moments. The long study duration supports the effects of the intervention on a longtime basis.
The efficacy of "LBMI-A" (59), "TeleCoach", and "Ray's Night Out" (60) was less convincing. The poor methodological quality, the short study duration as well as the lack of statistical power for "LBMI-A" impaired the quality of the study and did not allow relevant conclusions to be drawn. As for "TeleCoach", the exclusive decrease in the frequency of use has no clinical value. The reduction in the quantity of use for "Ray's Night Out", only at 1 month, was attributed, by the authors, to an assessment effect where an unconscious change in habits of use might occur at inclusion. No plausible argument supports the effect of those applications on behavior change.
For other substances, "S-Health" tested on patients with opioid use disorders showed a positive impact on the reduction of use. However, biological verification did not correlate these results at 1-month assessment. This could be in part explained by the detection method and the duration of the study. Change in use behavior could also have been overestimated by memory bias due to retrospective self-report of use and randomization bias.
## Critical analysis of ineffective applications
Fourteen applications [9 for tobacco, 4 for alcohol, and 1 for BED] were considered to be ineffective. However, "Crush the crave" targeting smoking, which did not show greater efficacy than the manual guide, had a high quit rate (230/1599). Also, 30 more participants smoked less than one pack per day while using the application. Several factors may have influenced these results such as the frequency of use (higher for the guide) or the on-demand solicitation of the application which may be perceived as a burden by the user. The clinical impact of these findings is substantial. Moreover, the mechanism of delivery being different, the mobile application can be more easily disseminated than a paper booklet.
The use of BAC in the 'Promillekoll' application showed an unexplained increase in alcohol use. The calculation of BAC so as to limit use, in various studies, has not been proven effective.
Several biases have been identified in the other trials making the results on efficacy difficult to interpret, such as confusion factors with the control groupsor attrition biasbetween the two arms, the short study duration (less than 6 months)or the low statistical power of some trialswhich lead to an under-estimation of the results.
## Craving management
Twelve applications managed craving. "A-CHESS, " which showed probative results on change in use, specifically monitored craving and proposed real-time solutions to manage this symptom . Even if, "Craving to Quit" (47) and "SmartQuit" (43) did not show significant effect on use, their craving management techniques seemed effective, on the shortterm, on the intensity of craving, the association of craving and use or a better acceptance of the symptom which was correlated to a higher prevalence of abstinence. The lack of impact of physical activities on craving for the "PhoS" application could be explained by the fact that both study arms received information on the benefits of physical activities prior to the test (confusion factor).
The other applications did not systematically evaluate the impact of the interventions on craving which made it difficult to determine whether the craving management techniques, used in those applications, are really effective.
# Validity of results
Two applications (out of 8) which were considered effective by the authors had poor methodological qualitydue to inappropriate randomization. The JADAD score, used in this review, had certain limits. Double-blinding was not always possible between the study groups due to heterogeneous comparators (e.g., application compared to a paper booklet or no intervention). Some information could have been omitted by authors due to volume restriction of journals. Applications which are considered effective in one population, in a particular country should be tested in other contexts before any conclusion can be made on generalizability.
## Prospects of improvement for applications
More randomized controlled trials (RCTs) are required over a sufficiently longer period, minimum 12 months, and on a larger scale to be able to predict sustainable results. Information and monitoring are important features of health applications, however, the active involvement of the user (e.g., by daily tasks) could be more effective in enhancing the effects of the intervention. Mobile health interventions should continue to target the psychological mechanisms implied in behavior change, such as self-efficacy. A more systematic consideration of craving by the applications should be considered. The lack of support for craving might explain the failure to maintain change in behavior observed for some applications. The impact of these interventions must be measured in different contexts (with or without treatment, on different severity of addiction, in various sociodemographic contexts) to better understand their limitations and the profile of patients who could be more receptive to this type of intervention.
# Limitations
Some limitations of this review are to be acknowledged. First, searches were led in only three databases. Every effort was made to ensure that this review of the literature was comprehensive and encompassed all available and relevant literature however. Articles published elsewhere will not have been considered, however, we searched comprehensive databases for articles with the best methodological qualities. Articles not published in English or French language would have been missed by the search methodology. Secondly, selected applications are recent and further studies on their impact are in progress. Thirteen trials concerning addiction recovery applications have been identified in the Clinical Trial database. This systematic review highlights the current state of knowledge among heterogeneous data and questions remaining to be investigated.
# Conclusion
The current findings suggest that smartphone applications can effectively contribute to behavior change and craving management in SUDs and addictive disorders. However, to date, very few applications have been evaluated for validity. The evidence on the efficacy of mHealth addiction recovery applications are too limited at this time to be able to recommend them as an autonomous or complementary tool for the treatment of addictions. However, there is a signal that such applications could become significant contributors to treatment in the future. For that more rigorous RCTs including more homogeneous comparators are required, on larger scale and with longer-term evaluations so as to clarify the sustainability of the change in use behavior. Real-time interventions have immediate impact on behavior change. However, the long-term challenge is the prevention of relapse through the management of craving and global care. In that perspective, further research on mHealth is needed.
# Data availability statement
The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s. |
Multiplex real-time PCR for the detection of Clavibacter michiganensis subsp. michiganensis, Pseudomonas syringae pv. tomato and pathogenic Xanthomonas species on tomato plants
A multiplex real-time PCR method based on fluorescent TaqMan® probes was developed for the simultaneous detection of the tomato pathogenic bacteria Clavibacter michiganensis subsp. michiganensis, Pseudomonas syringae pv. tomato and bacterial spot-causing xanthomonads. The specificity of the multiplex assay was validated on 44 bacterial strains, including 32 target pathogen strains as well as closely related species and nontarget tomato pathogenic bacteria. The designed multiplex real-time PCR showed high sensitivity when positive amplification was observed for one pg of bacterial DNA in the cases of Clavibacter michiganensis subsp. michiganensis and Pseudomonas syringae pv. tomato bacteria and 100 pg for bacterial spot-causing xanthomonads. The reliability of the developed multiplex real-time PCR assay for in planta detection was verified by recognition of the target pathogens in 18 tomato plants artificially inoculated by each of the target bacteria and tomato samples from production greenhouses. OPEN ACCESS Citation: Peňázová E, Dvořák M, Ragasová L, Kiss T, Pečenka J, Čechová J, et al. (2020) Multiplex real-time PCR for the detection of Clavibacter michiganensis subsp. michiganensis, Pseudomonas syringae pv. tomato and pathogenic Xanthomonas species on tomato plants. PLoS ONE 15(1): e0227559. https://doi.org/10.wounds and natural openings such as hydathodes and stomata; however, plants may be infected from infested seeds. Once inside the plant, the pathogen proliferates in xylem vessels, forming extensive biofilm-like structures, which aid in colonization and movement through the plant. Moreover, bacterial ooze from cankers and hydathodes, spread by rain or irrigation water, causes rapid spread to nearbyplants[7][8][9]. The symptoms of the disease occur on aerial parts and include wilting of leaves and discoloration of vascular tissues resulting in stem canker. At the late stage of infection, black spots with white halos, so-called "bird eyes", may occur on fruit[10,11].Bacterial speck of tomato caused by Pst (Proteobacteria; family Pseudomonadaceae; genus Pseudomonas) is one of the most persistent bacterial diseases in both the greenhouse and field production of tomatoes. The disease is spread by contaminated tomato seeds and infected weeds in which the bacteria can survive in the weed root system[12,13]. The pathogen enters the intercellular spaces of leaves through stomata or the base of leaf trichomes and multiplies endophytically and asymptomatically prior to symptom development. The disease is characterized by small necrotic lesions surrounded by chlorotic haloes on the leaves, stems and fruits of tomato plants[5,6,14].Bacterial spot of tomato is caused by multiple species of the genus Xanthomonas (Proteobacteria; family Xanthomonadaceae), most recently separated into four distinct species: X. euvesicatoria (Xe), X. vesicatoria (Xv), X. perforans (Xp) and X. gardneri (Xg)[15]. However, a close evolutionary relationship between Xe and Xp was reported[16,17]. Among the four species, Xe, Xv and Xg infect both pepper and tomato, while Xp has only been reported on tomato[15]. In 2010, the strain Xp was also isolated from diseased pepper plants by Schwartz et al. [18]. Symptoms on tomato leaves are similar to those described for Pst. Small circular lesions sometimes with a yellow halo develop on the leaflets or on the edge of leaves. On immature fruits, dark green to black lesions are formed[19]. The disease is spread through infected seeds, irrigation water, wind or by infected plant debris[20,21]. Bacteria enter the host plants through stomata on the leaf surfaces or through wounds and colonizethe vascular system[22].Because of the economic importance of these pathogens, the accurate detection of causal organisms is crucial, and it also represents one of the most effective strategies to prevent their further spread[3,23]. For pathogen detection, PCR-based methods are the most commonly used. Many conventional PCR protocols for Cmm, Pst and BSX detection were described previously [3, 24-28], as well as real-time PCR assays [29-32] that increased the specificity and sensitivity of PCR detection. Additionally, the use of multiplex reactions has increased the efficiency of laboratories and has led to the faster and more effective identification of the causal organism. In the case of tomato pathogens, conventional multiplex PCR detecting Cmm, Pst and X. axonopodis pv. vesicatoria was described by Ö zdemir [1]. However, this reaction was optimized on pure bacterial cultures only when bacterial strains ICMP 2550 (Cmm), ICMP 2844 (Pst) and ICMP 9592 (Xe) were tested. The verification of designed multiplex assay on broader spectrum of isolates and diseased plants as well as a possible cross-reaction with nontarget bacteria was not shown. The use of previously described system for Cmm detection through pat-1 gene carried by plasmid pMC1 [33 bears also the risk of false negative results for isolates lacking this plasmid. Moreover, the separation of PCR products by gel electrophoresis requires more time and higher workload. According to our knowledge, the multiplex real-time PCR for detection of bacteria Cmm, Pst and BSX complex was not described up to now.The goal of this study was to design a multiplex real-time PCR assay based on TaqMan1 probes to achieve a rapid, sensitive and highly specific protocol for in planta detection of Cmm, Pst and BSX.Multiplex real-time PCR detection of tomato bacterial pathogens PLOS ONE | https://doi.org/10.
# Introduction
Clavibacter michiganensis subsp. michiganensis (Cmm), Pseudomonas syringae pv. tomato (Pst) and bacterial spot-causing xanthomonads (BSX) represent major bacterial pathogens of tomato. Cmm and BSX are quarantine organisms in the European Union (EPPO A2 list) and are subjected to strict international phytosanitary controls. However, significant losses caused by Pst have also been reported.
Cmm (Actinobacteria; family, Microbacteriaceae; genus, Clavibacter), the causal agent of bacterial wilt and canker of tomato, is considered one of the most important bacterial pathogens in tomato plantings worldwide. The pathogen infects tomato plants epiphytically through PLOS ONE | https://doi.org/10.1371/journal.pone.0227559 January 7, 2020 1 / 15 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111
# Materials and methods
## Bacterial strains and plant samples
Bacterial cultures (listed in S1 and S2 Tables) were obtained from the National Collection of Plant Pathogenic Bacteria (NCPPB, London, UK), National Collection of Agricultural and Industrial Microorganisms (NCAIM, Budapest, Hungary), Collection of University of Warwick (HRIW, Wellesbourne, UK) and Crop Research Institute (CRI, Prague, Czech Republic). In total, 44 pathogenic bacterial strains with different geographical origins were tested. The set of samples included 32 strains of target bacteria (six strains of Cmm, eight strains of Pst, 18 strains of BSX), 10 closely related species and two nontarget bacterial pathogens of tomato. The isolates were grown on Mueller-Hinton Agar (HiMedia, Mumbai, India) at 25˚C (genus Pseudomonas) and 28˚C (genera Clavibacter and Xanthomonas) for 2-3 days prior to DNA extraction. Positive controls for in planta detection were obtained from artificially inoculated tomato seedlings (Solanum lycopersicum, cv. Mandat) grown in isolated greenhouse conditions. Reference strains of Cmm (NCPPB 2979), Pst (NCPPB 1106), Xe (NCPPB 2968), Xv (NCPPB 422), Xg (NCPPB 881) and Xp (NCPPB 4321) grown overnight in Mueller-Hinton Broth (HiMedia, Mumbai, India), and bacterial suspensions of approximately 10 8 CFU.ml -1 were prepared in 0.9% sterile physiological saline solution. For each pathogen, 10 one-month-old seedlings were artificially inoculated. The bacterial suspension of Cmm (0.01 ml) was injected into the tomato stems using a sterile needle and syringe, and the suspension of Pst and each of the BSX pathogens were sprayed on the leaf surface using a hand atomizer (BOSCH PFS 55, Bosch, Germany). After the development of symptoms, three plants inoculated with each bacterium were randomly selected; symptomatic parts were used for DNA extraction and pathogen detection. As a negative control, the genomic DNA from healthy plants of seven tomato cultivars (Pedro, Palava, Sonet, Mandat, Curranto, Charmant and Gallant) was used. Seeds and seedlings of all tomato cultivars were provided by the company Moravoseed CZ a. s.
## Dna extraction
Total genomic DNA of bacterial cultures and DNA of tested plants was extracted with the NucleoSpin Tissue kit (Macherey-Nagel, Düren, Germany) according to the manufacturer´s instructions. The DNA of plant samples was isolated from approximately 100 mg of homogenized plant tissue. The DNA concentration of the samples was estimated with a Modulus™ Single Tube Multimode Reader (Turner BioSystems, CA, USA) and adjusted to a final concentration of 4-5 ng.μl -1 for bacterial cultures and 50 ng.μl -1 for the plant samples. A concentration of 50 ng.μl -1 was used for nontarget bacterial cultures to increase the probability of their detection in case of nonspecific amplification and to prevent possible false positive results.
## Primer and probe design
Primers and probeswere designed using Primer-BLASTand CLC Main Workbench 6.5 (CLC Bio, Aarhus, Denmark) software. The detection system for Cmm used the previously published TaqMan1 probe CMM-TP targeting the region of 16-23S rRNAthat allows the differentiation of Cmm from other Clavibacter michiganensis subspecies. Newly designed Cmm primers were constructed to be fully compatible with the Cmm probe. For the detection of Pst, the target region of RNA polymerase sigma factor hrpL (hypersensitivity and response pathogenicity) was used based on its involvement in pathogenicity. The hrp gene cluster is considered essential for symptom formation in host plants and for hypersensitive responses in nonhosts. The GTP-binding membrane protein lepA (hypothetical protein for elongation factor 4) was used as the target for BSX. LepA promotes the back translocation of tRNAs on the ribosome during the elongation cycle and has a high conservation in certain bacteria, thus allowing highly specific detection. All primers were tested for individual specificity by in silico analysis using a Blastn search (GeneBank/NCBI, BLAST 2.2.31+) in which nonredundant collection (nr/nt) and highly similar sequences (megablast) settings were used. The self-complementarity, primer-dimer estimation and melting temperatures of each oligonucleotide were evaluated using Multiple Primer Analyzer (https://www.thermofisher. com/cz/en/home/brands/thermo-scientific/molecular-biology/molecular-biology-learningcenter/molecular-biology-resource-library/thermo-scientific-web-tools/multiple-primeranalyzer.html) and OligoAnalyzer Tool (https://www.idtdna.com/pages/tools/oligoanalyzer). For multiplexing, the following combinations of fluorophores and quenchers were used: FAM-BHQ1 (Pst), HEX-BHQ1 (Cmm) and Cy5-BHQ2 (BSX).
## Optimization of simplex real-time pcr assays
Detection systems were first optimized as simplex assays using DNA from isolates from the NCPPB collection (S1 and S2 Tables) and seven healthy cultivars of tomato. All samples were tested in triplicate on the Roche LightCycler 480 Instrument II (Roche Diagnostics, Basel, Switzerland). The reactions consisted of 10 μl of LightCycler 480 Probes Master (Roche Diagnostics Corp., Basel, Switzerland), 1.2 μl of each of the forward and the reverse primer (10 μM), 0.24 μl of probe (10 μM) and 2 μl of DNA template. The final reaction volume of 20 μl was achieved by the addition of nuclease-free water (Ambion, TFS, Waltham, USA). Annealing temperatures of 60, 62 and 65˚C were compared to optimize the thermal profile. The best combination of specificity and reaction efficiency was obtained with an annealing temperature of 65˚C. The thermal profile of the reaction was set to 95˚C for 10 min followed by 40 cycles of 95˚C for 10 s, 65˚C for 30 s and 72˚C for 1 s. A plate read was set to occur during the 72˚C cycle. The thresholds were set automatically according to manually selected noise bands within the assays for each TaqMan1 probe.
## Evaluation of assay sensitivity
The sensitivity of the simplex assays was evaluated on serial dilutions of DNA isolated from cultures of Cmm (NCPPB 2979), Pst (NCPPB 1109) and Xe (NCPPB 2689) reference strains. Only Xe was chosen from the BSX complex due to its prevalence in diseased tomatoes. DNA samples were diluted to approximately 50 ng.μl -1 with nuclease-free water, and then 10-fold dilution series (5×10 1 -5×10 −4 ng.μl -1 ) were prepared. All samples were tested in triplicate.https://doi.org/10.1371/journal.pone.0227559.t001
[formula] PST-hrpL_e_rev GATGCCCCTCTACCTGATGA PST-hrpL_TP GCTGAACCTGATCCGCAATCAC (FAM -BHQ1) XE_lepA_aec_fwd TGATCATCGATTCCTGGT lepA 197 bp XE_lepA_cea_rev GTTGATCCAGCCCACTTC XE_lepA_TP CCAGCGAGACCACGCCCA (Cy5-BHQ2) � Designed in [/formula]
TaqMan1 real-time PCR assays were performed on the Roche LightCycler 480 Instrument II (Roche Diagnostics Corp., Basel, Switzerland) using the mastermix LightCycler 480 Probes Master (Roche Diagnostics Corp., Basel, Switzerland) and the thermal profile as described above for the simplex assay optimization. The efficiency of each reaction was determined by a standard curve calculated with qPCR cycler software (LightCycler1 480 Software, Version 1.5.1.62). For the absolute quantification, the fit points method analysis module was used for all the analyses.
## Specificity of the multiplex real-time pcr assay and sensitivity analysis
Once the simplex reaction conditions were optimized, the three assay reactions were multiplexed into one. The triplex assay was performed on the Roche LightCycler 480 Instrument II using 96 multiwell plates. The reaction mixes were comprised of 10 μl of LightCycler 480 Probes Master (Roche Diagnostics Corp., Basel, Switzerland), 1.2 μl of each of the forward and reverse primers (10 μM), 0.24 μl of each probe (10 μM) and 2 μl of DNA template. The final reaction volume of 20 μl was achieved by the addition of nuclease-free water (Ambion, TFS, Waltham, USA). The thermal profile of the reaction was set to 95˚C for 10 min followed by 40 cycles of 95˚C for 10 s, 65˚C for 30 s and 72˚C for 1 s. A plate read was set to occur during the 72˚C cycle. All samples were tested in triplicate. The multiplex assay was verified on 44 isolates, which are listed in the S1 and S2 Tables. The sensitivity of the assay was evaluated using the same serial dilutions of target bacterial DNAs as in simplex sensitivity analyses.
## Mixed template experiment
Template DNA from Cmm (NCPPB 2979), Pst (NCPPB 1109) and Xe (NCPPB 2689) were mixed together in a 1:1:1 ratio (50 ng.μl -1 starting concentrations), and the 10-fold dilution series in the range of 5×10 1 -5×10 −4 ng.μl -1 were tested in simplex and triplex real-time PCR assays. Three replicates of each dilution were used. Briefly, leaves suspected for pathogen presence were surface sterilized with 0.5% sodium hypochlorite and four pieces with approx. sizes of 1 cm 2 were placed into 1 ml of physiological solution (0.9% NaCl) for 15 min. For plating on media, 50 μl of the extract was used.
## Testing the assay on plant samples from commercial greenhouses
# Results
## In silico analysis of primers and probe specificity
Oligonucleotide primers were first tested for formation of self-dimers and primer-dimers. According to the Multiple Primer Analyzer tool, self-dimers do not form. The possible primer-dimers are presented in S1 Fig. The specificity of designed primers was tested by in silico analysis using a Blastn search (GenBank/NCBI). Designed sequences were compared with sequences available in the GenBank database using a threshold of 95% similarity. The organisms possibly amplified by the designed oligonucleotides are presented in S3The in silico analyses showed that the combination of oligonucleotides for Cmm and Pst detected only the target organisms. The primers and probe used for BSX detection showed the possible amplification of five other bacteria, Xanthomonas axonopodis pv. commiphorae, Xanthomonas campestris pv. arecae, Xanthomonas campestris pv. musacearum, Xanthomonas fuscans subsp. fuscans and Xanthomonas vasicola pv. vasculorum. However, these pathogens have not been described on tomato plants.
Designed sequences were also compared with partial or complete genome sequences of target bacteria available in GenBank. The primers and probe designed for Cmm detection had
## Specificity and sensitivity of simplex real-time pcr
Detection systems were tested on DNA from the isolates listed in S1 and S2 Tables and seven healthy cultivars of tomato. At the annealing temperature of 65˚C, only strains X. axonopodis pv. phaseoli and X. hortorum pv. carotae showed nontarget amplification in the assay for BSX detection (S4 .
The sensitivity of the simplex reaction was evaluated on serial dilutions of genomic DNA purified from Cmm (NCPPB 2979), Pst (NCPPB 1106) and Xe (NCPPB 2689). The
## Specificity of multiplex real-time pcr assay
The multiplex assay was tested on the bacterial isolates listed in S1 and S2 Tables. The specificity of the designed primers and probes was tested on 32 target bacteria,The assay for Pst identification (FAM channel) successfully amplified seven out of eight Pst isolates, although detectable fluorescence was not recorded for Pst strain NCPPB 878. The sequence amplified from NCPPB 878 with hrpL primers in conventional PCR showed variability at the hybridization site of the TaqMan1 probe in two positions where a nucleotide substitution of T instead of C was present in the Pst reference sequence (NC_004578). Nonspecific amplification was not observed for the other bacteria.
Primers and the probe designed for the detection of the lepA gene of BSX pathogens (Cy5 channel) proved reliable for identification of all BSX members, X. euvesicatoria, X. vesicatoria, X. gardneri and X. perforans, as well as for the X. axonopodis pv. vesicatoria strains. Nonspecific amplification was observed only in the case of X. axonopodis pv. phaseoli and X. hortorum pv. carotae. The reactions with the other bacteria showed negative results.
No amplification occurred from all samples of plant genomic DNA extracted from healthy tomato plants.
## Sensitivity of the designed multiplex real-time pcr assay
The sensitivity of the multiplex real-time PCR exhibited consistent amplification of the 16S rRNA and hrpL genes from 1 pg of genomic DNA in the reaction. The sensitivity of detection was 100 times lower for the of lepA gene compared to the 16S rRNA and hrpL genes. The reaction efficiency reached values of 97.8% for Cmm, 95.5% for Pst and 99.3% for BSX detection.
## Mixed template experiment and in planta detection
The DNA of Cmm, Pst and Xe mixed at a ratio of 1:1:1 was diluted in a 10-fold dilution series and tested by the real-time PCR assay. Positive results were obtained up to the 1000-fold dilution in both simplex and multiplex assays, which represented the amount of 1 pg of genomic DNA in the reaction. In the case of the 1000-fold dilution, the Cmm (HEX) and Pst (FAM) detection exhibited mean Ct values of approximately 30 in both the simplex and multiplex reactions. The Ct values for Xe detection were higher than those obtained for Cmm The in planta detection of the designed assay was verified on artificially inoculated onemonth-old tomato plants. The target pathogens were successfully detected by multiplex realtime PCR assay using 16-23S rRNA, hrpL and lepA genes on appropriate channels in all tested samples.
## Testing the designed multiplex assay on greenhouse samples
Leaf samples of four tomato cultivars randomly collected from plants in production greenhouses were tested for the presence of pathogenic Cmm, Pst and BSX by the designed multiplex real-time PCR assay. None of the 20 samples showed positive detection of the target organisms. The presence of pathogens was also tested by cultivation of leaf extracts on selective media, but characteristic colonies were not observed.
# Discussion
This study provides the first report of a multiplex TaqMan1 real-time PCR assay that simultaneously targets the Cmm, Pst and BSX complex. For the detection of these pathogens, conventional multiplex PCR was optimized by Ö zdemirusing previously published systems for Cmm, Pst detection. However, this system was tested only on three cultures, Cmm ICMP 2550 (NCPPB 2979), Pst ICMP 2844 (NCPPB 1106) and Xav ICMP 9592, which were recently classified as X. euvesicatoria. Additionally, the specificity analyses were confirmed neither on a broader spectrum of target and nontarget isolates nor on plant samples. Because Cmm and BSX are quarantine organisms, the use of real-time PCR methods is more accurate for providing information about the health of tested material. Real-time PCR assays using TaqMan1 probes for Cmm detection were previously published. Available detection systems use the target sequence of the internal transcribed spacer (ITS) located between the 16S and 23S rDNA, as in this study. This region is generally used to avoid possible false-negative results that can be reported when plasmidborne genes encoding pathogenicity (pat-1 and celA) are targeted. The specificity of the designed assay for Cmm was confirmed by the specific detection of C. michiganensis and. Nevertheless, based on the nonspecific amplification of Pectobacterium carotovorum subsp. carotovorum and Pst isolate CRI 211, the cycle cutoff value of Ct 37 is recommended. The application of a cut-off value in qPCR protocols is frequently used by diagnostic laboratories to prevent false positive results. The qPCR system targeting putative two-component system sensor kinasesfor Cmm identification that was recommended by EPPOalso applies the limit of Ct 35. This protocol was not used in this study because of the possible hybridization of the primers RZ_ptssk 10/RZ_ptssk 11 with Pst isolates, as a difference in two nucleotides between both primers and the Pst reference sequence was found by in silico analysis.
The designed system for the identification of Pst pathogens targets the hrpL gene from the hrp cluster, which was reported as suitable for PCR-specific detection. The hrpL gene encodes the alternative sigma factor required for the expression of the hrp gene cluster in the Pseudomonas syringae group, which is essential for symptom development in host plants and the hypersensitive response in nonhosts. The specificity of the reaction was proven for seven of eight tested isolates of Pst. Nonspecific amplification was not observed. In the case of strain NCPPB 878, lack of detection was caused by nucleotide substitutions at the probe hybridization site. However, the nucleotide sequence of the target locus of the hrpL gene showed 97% identity with Pseudomonas syringae pathovars aptata, panici, pisi and syringae but only 88% identity with the Pst reference sequence (GenBank Acc. No. NC_004578) when the Blastn search (BLAST 2.2.31+) was applied. Additionally, the NCPPB collection does not declare the authenticity of isolate 878, although the origin from S. lycopersicum and its pathogenicity was confirmed by NCPPB.
The published real-time PCR protocols for BSX pathogens are focused mainly on the distinction of the involved species. Since the worldwide distribution of X. perforansand X. gardneriwas reported, the presence of all BSX species should be tested. In the detection of the BSX group, the lepA gene was successfully amplified from all four species and natural isolates from the CRI collection. Nonspecific amplification was obtained only for the isolates X. axonopodis pv. phaseoli and X. hortorum pv. carotae. Based on the in silico analyses of the designed oligonucleotides, the amplification of X. a. pv. phaseoli should be prevented by incompatibility with the BSX probe. Nevertheless, none of these pathogens were reported on tomato plants; thus, a false positive result is not expected.
In sum, the designed assay proved high specificity in both simplex and multiplex reactions. The specificity of the multiplex assay was not influenced by the involvement of the primers and probes used except for Pectobacterium carotovorum subsp. carotovorum and Xanthomonas axonopodis pv. phaseoli, which were detected by the assay designed for BSX. The reliable results were also obtained when target DNAs were mixed and 1000 times diluted to the final amount of approx. 0.1 ng of genomic/total DNA per reaction. The verification of plant samples confirmed the usability of the designed detection assay, which allows the recognition of quarantine organisms. The negative result for samples from commercial greenhouses confirmed the high level of phytosanitary practice that is required for the ecological approach to be applied by concerned companies. The verification of the designed assay on seeds was not performed based on the quarantine character of pathogenic Cmm and BSX and strict sanitary controls that created the unavailability of infected seeds for this study. Nevertheless, this multiplex real-time PCR based on three TaqMan1 probes was shown to be a useful tool for the quick, specific and sensitive detection of the quarantine and economically important tomato bacterial pathogens Clavibacter michiganensis subsp. michiganensis, Pseudomonas syringae pv. tomato and bacterial spot-causing xanthomonads in both bacterial cultures and tomato plants.
Supporting information S1 for primer dimer detection.
(TIF) S1 |
Triple Intussusception in an Adult—A Rare Presentation of Adenocarcinoma Ileum
Intussusception involves the telescoping of a segment of bowel into an adjacent segment. This leads to complications such as obstruction, inflammation, and possible ischemia. Intussusception is the leading cause of intestinal obstruction in children. However, adult intussusception is rare, accounting for less than 5% of all bowel obstructions and 5% of all intussusceptions. 1,2 Simultaneous occurrence of multiple intussusceptions is a rare condition, exact incidence of which is still unavailable.3Intussusceptions in children are typically primary or idiopathic. In contrast, most adult intussusceptions have a demonstrable etiology in 70 to 90% of the cases. 4 Sixty-six per cent of colonic intussusceptions and 30% of cases in the small intestine have malignant neoplasms as their lead point.Adenocarcinoma is the most common malignant lead point in the colon, whereas metastasis is the most common in the small intestine.5,6Small bowel neoplasms are exceedingly rare, constituting only 5% of all gastrointestinal neoplasms and only 1 to 2% of all gastrointestinal malignant tumors, despite the fact that they comprise 90% of the length of the alimentary tract. 7,8 Malignant neoplasms of the small bowel are primarily of two etiologies: neuroendocrine tumors which account for $40% of cases and adenocarcinoma which account for another 40%. The remaining 20 to 25% comprise gastrointestinal stromal tumors, sarcomas, and lymphomas. 9 In descending order of frequency, small bowel adenocarcinoma may occur within the duodenum (48.4%), jejunum (32.5%), or ileum (19.2%). 10KeywordsAbstractIntestinal intussusception is uncommon in adults. It occurs more often in the small intestine than in the colon. In adults, when small bowel intussusception occurs, it can be due to a malignant lead point. Malignant etiology is most frequently due to diffuse metastatic disease. We present a rare case of an 18-year-old woman who was diagnosed with jejunojejunal, jejunoileal, and colocolic intussusceptions. She presented with vomiting, abdominal pain, and passage of semisolid stools for 5 days. During emergency exploratory laparotomy, multiple polyps were found in the jejunum, ileum, and sigmoid. Jejunotomy and sigmoidotomy were done to remove the respective polyps. The ileal polyp showed hemorrhagic changes; hence, an intraoperative decision was taken to proceed with resection and anastomosis. On histopathological examination, the resected ileal part showed moderately differentiated adenocarcinoma (grade 2) arising from an adenomatous polyp, while the jejunal polyp and sigmoid polyp were adenomatous polyps with low-grade dysplasia. Patient received six cycles of adjuvant chemotherapy consisting of capecitabine and oxaliplatin (CAPEOX regimen). After 2 years, she is symptom free with a normal colonoscopy. The treatment of intussusception in adults typically involves surgery, often with bowel resection as there is always a pathologic leading cause which may be malignant, like in our case.
Intussusception involves the telescoping of a segment of bowel into an adjacent segment. This leads to complications such as obstruction, inflammation, and possible ischemia. Intussusception is the leading cause of intestinal obstruction in children. However, adult intussusception is rare, accounting for less than 5% of all bowel obstructions and 5% of all intussusceptions. 1,2 Simultaneous occurrence of multiple intussusceptions is a rare condition, exact incidence of which is still unavailable. [bib_ref] Simultaneous multiple intussusceptions in an adult patient caused by multifocal non-Hodgkin's lymphoma:..., Chamarthi [/bib_ref] Intussusceptions in children are typically primary or idiopathic. In contrast, most adult intussusceptions have a demonstrable etiology in 70 to 90% of the cases. [bib_ref] Adult intussusception, Azar [/bib_ref] Sixty-six per cent of colonic intussusceptions and 30% of cases in the small intestine have malignant neoplasms as their lead point.
Adenocarcinoma is the most common malignant lead point in the colon, whereas metastasis is the most common in the small intestine. [bib_ref] Clinical spectrum and surgical approach of adult intussusceptions: a multicentric study, Barussaud [/bib_ref] [bib_ref] Adult intussusception, Lu [/bib_ref] Small bowel neoplasms are exceedingly rare, constituting only 5% of all gastrointestinal neoplasms and only 1 to 2% of all gastrointestinal malignant tumors, despite the fact that they comprise 90% of the length of the alimentary tract. [bib_ref] A rare case of small bowel adenocarcinoma presenting as multiple intussusceptions: an..., Gupta [/bib_ref] [bib_ref] The clinical pathological features, diagnosis, treatment and prognosis of small intestine primary..., Guo [/bib_ref] Malignant neoplasms of the small bowel are primarily of two etiologies: neuroendocrine tumors which account for $40% of cases and adenocarcinoma which account for another 40%. The remaining 20 to 25% comprise gastrointestinal stromal tumors, sarcomas, and lymphomas. [bib_ref] Small bowel cancer in the United States: changes in epidemiology, treatment, and..., Bilimoria [/bib_ref] In descending order of frequency, small bowel adenocarcinoma may occur within the duodenum (48.4%), jejunum (32.5%), or ileum (19.2%). 10
## Keywords
[formula] ► adult intussusceptions ► adenocarcinoma ileum ► multiple intussusceptions [/formula]
# Abstract
Intestinal intussusception is uncommon in adults. It occurs more often in the small intestine than in the colon. In adults, when small bowel intussusception occurs, it can be due to a malignant lead point. Malignant etiology is most frequently due to diffuse metastatic disease. We present a rare case of an 18-year-old woman who was diagnosed with jejunojejunal, jejunoileal, and colocolic intussusceptions. She presented with vomiting, abdominal pain, and passage of semisolid stools for 5 days. During emergency exploratory laparotomy, multiple polyps were found in the jejunum, ileum, and sigmoid. Jejunotomy and sigmoidotomy were done to remove the respective polyps. The ileal polyp showed hemorrhagic changes; hence, an intraoperative decision was taken to proceed with resection and anastomosis. On histopathological examination, the resected ileal part showed moderately differentiated adenocarcinoma (grade 2) arising from an adenomatous polyp, while the jejunal polyp and sigmoid polyp were adenomatous polyps with low-grade dysplasia. Patient received six cycles of adjuvant chemotherapy consisting of capecitabine and oxaliplatin (CAPEOX regimen). After 2 years, she is symptom free with a normal colonoscopy. The treatment of intussusception in adults typically involves surgery, often with bowel resection as there is always a pathologic leading cause which may be malignant, like in our case.
We report a rare case of multiple simultaneous small bowel intussusceptions secondary to adenocarcinoma of the ileum in an 18-year-old woman. The statistics mentioned earlier emphasizes the rarity in reporting of these two conditions occurring together.
## Case report
An 18-year-old woman presented to the OPD with complaints of vomiting, abdominal pain, and passage of semisolid stools for 5 days. There was no history of fever, hematemesis, or malena. She did not have similar episodes in the past. On examination, she was vitally stable. She had diffuse tenderness and distension on per abdominal examination. Rest of abdominal examination was unremarkable. Per rectal examination was normal.
On investigating further, plain X-ray of the abdomen showed no abnormalities. Ultrasonography of abdomen and pelvis revealed: ileocolic intussusception in the right iliac fossa with ileum extending into ascending colon up to right half of transverse colon; ileoileal intussusception in left iliac fossa for a length of 8 cm; multiple enlarged mesenteric lymph nodes of average size 10 mm; and mild ascites. A contrast-enhanced computed tomography (CECT) of the whole abdomen confirmed the diagnosis demonstrating three regions of intussusceptions, namely, jejunojejunal, jejunoileal, and colocolic intussusceptions and irregular wall thickening involving the sigmoid colon (►Fig. 1).
After obtaining consent, patient was taken up for emergency exploratory laparotomy. All three intussusceptions were reduced without any complications. Bowel wall was mildly edematous without any obvious signs of ischemia/necrosis. Multiple polyps in the jejunum, ileum, and sigmoid colon were found to be lead points (►Fig. 2). Jejunotomy and sigmoidotomy were done to remove the respective polyps. The ileal polyp was hemorrhagic in appearance, which was suspicious of malignancy. Since frozen section was not available at the time of surgery, an intraoperative decision was made to proceed with ileal resection with 5 cm margin proximal and distal to polyp. The specimens were sent for histopathological examination. Rest of the abdomen was examined and found to be normal. The patient tolerated the surgery well. She made an uneventful recovery. Patient was discharged on postoperative day 5.
Histopathological report revealed: resected ileal segmentmoderately differentiated adenocarcinoma (grade II) arising on the background of adenomatous polyp; jejunal polyp-adenomatous polyp with low-grade dysplasia; sigmoid polyp-adenomatous polyp with low-grade dysplasia; and appendixchronic appendicitis (no granulomas/malignancy) (►Fig. 3).
On follow-up, patient was started on CAPEOX regimen (tablet capecitabine 500 mg 2-0-2 Â 14 days injection oxaliplatin 180 mg), 21-day cycles for 6 cycles. Patient responded well to the regimen. Positron emission tomography-CT done 3 months postoperatively showed fluorodeoxyglucose avid small benign polyp in anterior wall of rectum for which polypectomy was done. Patient is symptom free and doing well till date with regular follow-up of 2 years.
# Discussion
Intussusception is defined as the invagination of one segment of the bowel into an immediately adjacent segment. It can occur anywhere in the bowel. Intussusception in children usually occurs between 6 and 18 months of age. The incidence of intussusception declines with age accounting for only 1 to 5% of bowel obstructions in adults. [bib_ref] Intussusception of the bowel in adults: a review, Marinis [/bib_ref] [bib_ref] The diagnosis and management of adult intussusception, Begos [/bib_ref] [bib_ref] Intestinal intussusception: etiology, diagnosis, and treatment, Marsicovetere [/bib_ref] In the pediatric population, intussusceptions are typically primary or idiopathic with only 10% of cases having an identifiable precipitating lesion. In contrast, 70 to 90% of the cases of adult intussusceptions are caused by a structural lesion/lead point pathology which can be intraluminal, mural, or extramural. [bib_ref] Intussusception of the bowel in adults: a review, Marinis [/bib_ref] [bib_ref] The diagnosis and management of adult intussusception, Begos [/bib_ref] A lead point intussusception involving the small bowel is generally due to a benign condition. 12 Nonmalignant etiology includes benign tumors, adhesions, lymphoid hyperplasia, cystic fibrosis, scleroderma, celiac disease, inflammatory bowel disease, appendicitis, pancreatitis, and rectal foreign bodies. Sixteen per cent of small bowel and 5% of large bowel intussusceptions are idiopathic. [bib_ref] Adult intussusception, Lu [/bib_ref] Sixty-six per cent of colonic intussusceptions and 30% of intussusceptions in the small intestine occur due to malignant lead points. The most common malignant lead point in colon is adenocarcinoma and in the small intestine, it is diffuse secondary metastasis. [bib_ref] Adult intussusception, Lu [/bib_ref] [bib_ref] A rare case of small bowel adenocarcinoma presenting as multiple intussusceptions: an..., Gupta [/bib_ref] Our patient is an 18-year-old woman, who presented to us with multiple intussusceptions of small bowel due to adenocarcinoma of the ileum, both of which are rare entities.
Adults rarely present with the classic triad of abdominal pain, bloody currant jelly stools, and palpable tender abdominal mass seen in children. They may present as acute intestinal obstruction/perforation or more commonly chronic intermittent cramping abdominal pain associated with nonspecific signs of bowel obstruction. [bib_ref] The diagnosis and management of adult intussusception, Begos [/bib_ref] The nonspecific nature of these findings can result in a broad differential diagnosis.
Preoperative diagnosis ranges from 30 to 70%. 4,13 Evaluation often starts with X-ray of the abdomen which may reveal signs of intestinal obstruction or perforation but it lacks sensitivity and specificity for diagnosing intussusception. [bib_ref] Uncommon conditions in surgical oncology: acute abdomen caused by ileocolic intussusception, Mrak [/bib_ref] Ultrasound is both diagnostic and therapeutic with 100% sensitivity and specificity in children. [bib_ref] Ileoileal intussusception in children: ultrasonographic differentiation from ileocolic intussusception, Wiersma [/bib_ref] Nonoperative reduction of the intussusceptum by ultrasound-guided or fluoroscopic pneumatic or hydrostatic enema is successful in 85 to 90% of pediatric cases. [bib_ref] Ileocolic versus small-bowel intussusception in children: can US enable reliable differentiation?, Lioubashevsky [/bib_ref] However, ultrasound tends to be less accurate in adults but may still reveal classic features. In our patient, two out of three intussusceptions could be detected by ultrasound, but etiology could not be identified.
Abdominal CECT represents the most accurate diagnostic modality with the possibility of demonstrating lead points, vascular compromise, and possible associated complications such as intestinal obstruction or local spread. [bib_ref] Martínez de Alegría A. Intussusception in adults: what radiologists should know, Baleato-González [/bib_ref] Classic radiological findings include "target," "bulls-eye," or sausage-shaped lesions as a concentric hyperdense double ring, features owing to the anatomic configuration of the outer intussuscipiens and the central intussusceptum creating a bowel-within-bowel appearance. [bib_ref] Intussusception in adults: clinical characteristics, diagnosis and operative strategies, Yakan [/bib_ref] Multiple intussusceptions and irregular wall thickening involving the sigmoid colon could be identified on CECT in our patient.
The approach to management of intussusception is different in pediatric and adult intussusceptions. While in children, treatment is primarily based on radiological reduction, surgical resection is recommended in cases of adult intussusception because it is often associated with a lead point which can be malignant.
A selective approach to bowel resection is recommended when patient requires operative management, depending on Triple Intussusception in an Adult Jadhav, Krishnan e273 the age, pathology, and location of lead point. Bowel resection following the appropriate oncologic principles is recommended given the relatively high incidence of malignancy in adults. [bib_ref] Adult intussusception, Lu [/bib_ref] Curative resection is the treatment of choice and a chance for long-term survival. Lymphovascular invasion and positive surgical margins are predictors of locoregional recurrence and poor outcome. [bib_ref] Adenocarcinoma of the small intestine: 21-year review of diagnosis, treatment, and prognosis, Bauer [/bib_ref] In our patient, multiple dysplastic polyps could be found which warranted resection of bowel segment.
The suggested first-line adjuvant chemotherapy for adenocarcinoma of ileum is a combination regimen of 5-fluorouracil infusion or oral capecitabine with oxaliplatin. [bib_ref] The clinical pathological features, diagnosis, treatment and prognosis of small intestine primary..., Guo [/bib_ref] Other agents such as irinotecan and gemcitabine have also been tried. The use of biological agents and targeted therapy in patients with this rare tumor is under trial. [bib_ref] Small bowel cancer in the United States: changes in epidemiology, treatment, and..., Bilimoria [/bib_ref] Our patient was given six cycles of CAPEOX regimen. She is recurrence free till date.
# Conclusion
Adult intussusception is a rare entity and challenging to diagnose due to nonspecific symptoms with a vast differential diagnosis. Small bowel tumors are a rare but possible etiology of adult intussusception. Surgical exploration is the only way of making and confirming the diagnosis of small bowel adenocarcinoma despite the availability of sophisticated imaging modalities.
## Informed consent
Informed consent was obtained from the participant.
# Funding
None.
## Conflict of interest
None declared.
[fig] Figure 1: Contrast-enhanced computed tomography of the abdomen showing (A) jejunojejunal intussusception (marked with red arrow) and ileoileal intussusception (circled with red) in coronal section, (B) ileoileal intussusception (circled with yellow) in sagittal section, and (C) colocolic intussusception involving sigmoid (yellow arrow) in sagittal section. [/fig]
[fig] Figure 2: Intraoperative findings (A) ileoileal intussusception, (B) jejunal polyp, and (C) sigmoid polyp. The Surgery Journal Vol. 7 No. 4/2021 © 2021. The Author(s). [/fig]
[fig] Figure 3: Orientation of the resected specimen. The Surgery Journal Vol. 7 No. 4/2021 © 2021. The Author(s). [/fig]
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Assessing the Factors Associated With Iran’s Intra-Industry Trade in Pharmaceuticals
Background: Pharmaceutical industry is a sensitive and profitable industry. If this industry wants to survive, it should be able to compete well in international markets. So, study of Iran's intra-industry trade (IIT) in pharmaceuticals is essential in order to identify competitiveness potential of country and boost export capability in the global arena.Methods: This study assessed the factors associated with Iran's intra-industry trade in pharmaceuticals with the rest of the world during the 2001-2012 periods using seasonal time series data at the four-digit SITC level. The data was collected from Iran's pharmaceutical Statistics, World Bank and International Trade Center. Finally, we discussed a number of important policy recommendations to increase Iran's IIT in pharmaceuticals.Results:The findings indicated that economies of scale, market structure and degree of economic development had a significantly positive impact on Iran's intra-industry trade in pharmaceuticals and tariff trade barriers were negatively related to IIT. Product differentiation and technological advancement didn't have the expected signs. In addition, we found that Iran's IIT in pharmaceuticals have shown an increasing trend during the study period. Thus, the composition of Iran trade in pharmaceuticals has changed from inter-industry trade to intra-industry trade.Conclusions:In order to get more prepared for integration into the global economy, the development of Iran's IIT in pharmaceuticals should be given priority. Therefore, paying attention to IIT could have an important role in serving pharmaceutical companies in relation to pharmaceutical trade.
# Introduction
Pharmaceuticals are considered as one of the essential needs of human beings which are produced using high level of technology. For this reason in trade arena, pharmaceuticals have always enjoyed strategic importance and hence governments attempt to pay specific attention to its production and trade along with other basic goods.
The rate of profitability, high turnover and employment of expert work force besides creating economic prosperity, engagement and promotion of knowledge power as well as making countries interdependent have given prominence to the pharmaceutical industry [bib_ref] Trend analysis of the pharmaceutical market in Iran; 1997-2010; policy implications for..., Kebriaeezadeh [/bib_ref]. The value of world pharmaceutical market with a share of 14 percent of the world GDP was 954 billion dollars in 2012. Average growth in the global pharmaceutical market has been 5.5% in the last decade [bib_ref] The best answer is trade. Iran Trade Promotion Organization, International Trade [/bib_ref].. Health care level of a country depends mostly on the accessibility of pharmaceuticals, thus, pharmaceutical trade wants more consideration of policy makers. The world pharmaceutical trade has expanded significantly because of world pharmaceutical consumption and production has increased in the recent years [bib_ref] Pharmaceutical policy and market in Iran: Past experiences and future challenges, Davari [/bib_ref]. But Iran's pharmaceuticals constitute a small share of international trade.
Pharmaceutical industry is ranked the second largest industry following electronic industry. Large pharmaceutical companies try to greatly increase their property by spending a lot of money to produce new drugs. In fact, pharmaceutical industry is a sensitive and advantageous industry [bib_ref] Iran pharmaceutical market, Cheraghali [/bib_ref]. According to the Iranian food and drug department, the share of Iran's pharmaceutical industry is about 2.6 billion of the 1200 billion dollars, while the share of Turkey as one of Iran's neighbors is 6 billion dollars .
The countries that have appropriate positions in world's economy usually undergo insignificant changes when confronted with tension. These countries are paving the way for maintaining more facilitation in their pharmaceutical commerce. They have mainly realized the fact that what brings about survival among international competitors is not only the degree of a country's self-sufficiency, but also, and more importantly, the extent to which it strengthens its position in international markets .
Because of pharmaceutical industry needs a high level of technological abilities in production, it appears to be a capital-intensive industry. So, intra-industry trade results from production efficiency in large scale, product differentiation and imperfect competition in this section. Intra-industry trade helps countries find the real comparative advantages of their pharmaceutical products by comparing themselves with other countries based on production methods and technologies. As we are seeking a suitable model for manufacturing, export and import of pharmaceuticals in order to promote and expand our foreign ties, knowing IIT are of utmost importance [bib_ref] Pharmaceutical Industry and Trade Liberalization Using Computable General Equilibrium Model, Barouni [/bib_ref].
Iran has numerous relative advantages regarding the pharmaceutical manufacturing conditions including access to infinite energy resources, relative progress in pharmaceutical technologies, high potential to enter into Islamic countries' markets, relative experience in pharmaceutical manufacturing, and cheap and young expert work force. These advantages can play an effective and competitive role in the Iran's IIT [bib_ref] Iran pharmaceutical market, Cheraghali [/bib_ref].
In addition, Iran is increasingly becoming integrated into the global economy. Thus, achieving a successful trade performance in pharmaceutical industry is of utmost importance in Iran's economy. Paying attention to economic integration through removing barriers of bilateral trade, creating trade zones, customs unions and other preferential trade arrangements will result in implementation of common economic policies in countries that have close economic ties with Iran. So, the share of intra-industry trade and the total trade between Iran and these countries will increase. Furthermore, the desirable participation of pharmaceutical industry in trade and absorbing foreign exchange can introduce this sector as the engine of economic growth. Regarding the importance of intra-industry trade in promoting of non-oil exports, it is recommended to take the determinants of intra-industry trade into consideration while protecting and maintain the relative comparative advantages. Moreover, given the low level of Iran's IIT of pharmaceuticals, trade liberalization is likely to have significant adjustment costs that can be reduced by promoting IIT of this industry. Therefore, study of the factors associated with Iran's intra-industry trade (IIT) in pharmaceutical industry is necessary in order to identify competitive potential of country and enhance export ability in the global arena and can be considered in foreign trade planning as a strategic and decision-making tool.
In Iran, no study related with IIT of pharmaceutical industry was found. Though, there are many studies associated to different economic sectors some of which would be explained.
The study in China has been performed by [bib_ref] Chinese bilateral intra-industry trade: A panel data study for 50 countries in..., Zhang [/bib_ref] named Chinese Bilateral Intra-Industry Trade: A Panel Data Study for 50 Countries in the 1992-2001 periods. In this study, five intra-industry trade drivers including Foreign Direct Investment (FDI), geographical distance, economic size, trade openness and trade composition has been considered. They observed that these factors had significant influences on IIT [bib_ref] Chinese bilateral intra-industry trade: A panel data study for 50 countries in..., Zhang [/bib_ref]. The other study associated with the determinants of IIT has been carried by [bib_ref] Can Gravity Model Explain BIMSTEC's Trade?, Kabir [/bib_ref] in the 1995-2008 periods by Gravity model. They found that trade liberalization, economic size and FDI had positive effects on IIT, but geographical distance had negative impact [bib_ref] Can Gravity Model Explain BIMSTEC's Trade?, Kabir [/bib_ref].examined the extent and pattern of intra-industry trade among Central and Eastern European Countries and the European Union during the time 1993 to 2001 using the Grubel-Lloyd index. They found a significant increase in intra-industry trade due to positive effects of economic size, Foreign Direct Investment (FDI), and human capital but negative impacts of geographical distance. Similar results were obtained by [bib_ref] An econometric view on the estimation of gravity models and the calculation..., Egger [/bib_ref] , over the 1985 to 1996, who demonstrated that the market size and distance had positive and negative impact on IIT of OECD manufacturing, respectively [bib_ref] An econometric view on the estimation of gravity models and the calculation..., Egger [/bib_ref].
## Examined intra industry trade (iit) of iran's agricultural products by indices of grubel & lioyd and
Fontagn & Freudenberg during time period 1997-2003. The Results showed small but rising IIT for agricultural products of Iran. Particularly, the IIT of Iran's agricultural products increased from 2.73 in 1997 to 5.98 percent in 2003, reflecting comparative advantage have an important role in foreign trade of agricultural products in Iran. The study of found IIT between Turkey and other selected OECD countries for the period 1985 to 2000 was negatively related to distance in both final products and intermediate products [bib_ref] Determinants of intra-industry trade in final goods and intermediate goods between Turkey..., Türkcan [/bib_ref]. Investigation by [bib_ref] Patterns and determinants of intra-industry trade in Asia, Sawyer [/bib_ref] showed that IIT was positively related to income per capita of its trading partners. Also, removal of trade barriers had a positive impact on trade intensity [bib_ref] Patterns and determinants of intra-industry trade in Asia, Sawyer [/bib_ref].
# Methods
## Theory of intra-industry trade
Globalization of economy is the most important and transparent distinctive feature of today's economy. This means that borders are being opened, trade is promoted, and regional trade and economic unions and zones are expanded. Various criteria are used to measure and determine a country's trade potential and competitiveness. Intra-industry trade is a suitable criterion that can show the trade flows between the economic sectors of two countries and their trade relations. The common theories of international trade only consider the inter-industry trade (trade of different products associated with different industries) and use simple assumptions such as the existence of a perfect competitive market, homogeneity of goods and increasing returns to scale. Thus, a major share of trade that is associated with the simultaneous trade of goods in a certain industry remains unexplained. Therefore, in the late 1970s, the intra-industry trade theories gradually started to develop and become widespread. Such theories are based on assumptions including increasing returns to scale and imperfect competition.
Intra-industry trade is an empirical phenomenon that has been interred into international trade discussions in recent years. Intra-industry trade takes place when a country simultaneously exports and imports of goods or services which produced in the same industry. If goods are differentiated by their appearance or horizontal distinctions, we are dealing with what is known as horizontal intra-industry trade. If goods are differentiated by quality and numerical distinctions, the phenomenon is called vertical intra-industry trade [bib_ref] Does the measurement of intra-industry trade matter?, Gullstrand [/bib_ref]. This type of trade is distinct from inter-industry trade. In inter-industry trade, a country is specialized in the production and export of a certain good or service and exports it instead of a different product or service in which it does not have any comparative advantage. The intra-industry trade phenomenon is a very important issue in developing countries. Paying attention to this phenomenon helps countries become aware of their own trade potentials and enhance their trade ties with each other and other countries.
In summary, intra-industry trade is as a consequence of variation in consumer preferences and economies of scale which occurs under monopolistic or imperfect competition [bib_ref] The nature and causes of intra-industry trade: Back to the comparative advantage..., Blanes [/bib_ref].
## Measurement of iran's intra-industry trade in pharmaceuticals
The most commonly used measure of IIT proposed by Grubel and Lloyd. In this index, inter-industry trade is calculated as the ratio of the absolute difference between exports and imports (net trade) over the total trade of the industry. In other words, intra-industry trade is expressed as the ratio of the total trade remaining after subtraction of net trade over the total trade of the industry. In summary, the Grubel-Lloyd Index calculates intra-industry trade as the difference between inter-industry trade and one.
[formula] GL it = | | 100, or GL it = 1 | | 100(1) [/formula]
Where X it and M it denote the values of export and import of industry i in year t, respectively. The index includes bounded interval of zero and 100. The index of Grubel-Lloyd (GL i ) equals 100 if all trade is intra-industry trade (i.e. imports and exports of country from the same industry are equal) and if the extent of GL i is zero, the total trade is inter-industry trade and there is no intra-industry trade in that industry [bib_ref] Measurement of Intra-Industry Trade, Wei [/bib_ref].
## Major associated hypothesises (variables) with iran's intra-industry trade in pharmaceuticals with the rest of the world
On the basis of advanced theories of trade and previous empirical studies about intra-industry trade, the www.ccsenet.org/gjhs Global Journal of Health Science following elements used as explanatory variables in this analysis to measure Iran's intra-industry trade in pharmaceuticals with the rest of the world, these include: (1) product differentiation (PDIF i );
(2) economies of scale (ES i ); (3) Market Structure (MS i ); (4) degree of economic development (DD i ); (5) technological advancement (KL i ) and (6) trade barriers (TR i ). These factors are vitally important in the explanation of IIT in pharmaceutical industry that is chosen for in the analyses.
Hypothesis 1: a direct relationship exists between product differentiation and Iran's IIT in pharmaceuticals.
Pharmaceutical productions are research-oriented activities; product differentiation in pharmaceuticals (by style, quality or technology) will be measured by the ratio of R&D expenditures (RDX i ) to the value added (VA i ) in Iran's pharmaceutical products. If different countries have high technology in different sections of the same industry, the ratio has a positive impact on IIT, and if one country has a higher degree of technology in production than the others, the ratio of R&D expenditure to value added has a negative impact on IIT.
[formula] PDIF i = RDX i /VA i(2) [/formula]
It is assumed that an increase in research and development costs relative to value added will increase the versions of technologically differentiated pharmaceutical products. Thus, it will have a positive effect on the extent of intra-industry trade.
## Hypothesis 2: it is expected that a positive relationship exists among economies of scale and iran's iit in pharmaceuticals.
Since large economies can readily gain from economies of scale, they can make use of benefits resulting from their large domestic market easily and so, have higher extent of IIT [bib_ref] The Pattern and Determinants of Intra-Industry Trade in Australian Manufacturing, Sharma [/bib_ref]. The ratio of pharmaceutical turnover (total sales) (TUR i ) to the number of pharmaceutical companies (MS i ) are used to compute economies of scale (ES i ) for Iran's pharmaceutical industry, in this research.
[formula] ES i = TUR i /MS i(3) [/formula]
It is assumed that a positive relationship exists between economies of scale and intra-industry trade in pharmaceuticals.
## Hypothesis 3: market structure is positively related to the level of iran's iit in pharmaceuticals.
In theory, industries with a large number of companies manufacturing differentiated products are more likely to engage in a higher level of bilateral trade rather than those with only a few companies. The variety of differentiated pharmaceutical products will increase under imperfect competition when a large number of pharmaceutical companies enter the market [bib_ref] Vertical and horizontal intra-industry trade: A cross industry analysis for the United..., Greenaway [/bib_ref]. It is expected that the number of pharmaceutical companies (MS i ) have a positive effect on the level of intra-industry trade.
## Hypothesis 4: the relationship among iran's iit in pharmaceuticals and the degree of economic development is positive.
A high per capita GNP indicates that the income level is high. As per capita income grows, consumers will demand more of differentiated products in their bilateral trade [bib_ref] Determinants of trade: The case of Mexico, Ekanayake [/bib_ref]. In this study, Iran's per capita GNP is applied as a measure of the degree of Iran's economic development (DD i ) that would be expected to have a positive relationship with Iran's IIT in pharmaceuticals.
## Hypothesis 5: iran's iit in pharmaceuticals have a positive relationship with technological advancement.
The higher ratio of capital relative to labor force in a country result in more likely an increase in the production of differentiated products and increasingly engage in IIT [bib_ref] International trade in the presence of product differentiation, economies of scale and..., Helpman [/bib_ref]. Pharmaceutical production in a country also depends on the level of capital formation. The ratio of total capital to the labor force (KL i ) is used in this study to demonstrate Iran's technological advancement.
[formula] KL i = (K i /L i )(4) [/formula]
Where, K i and L i are the real capital formation and the total labor force of Iran. This ratio is assumed to have a positive relationship with IIT.
## Hypothesis 6: trade barriers have a negative effect on iran's iit in pharmaceuticals.
Eliminating trade barriers facilitates the development of IIT and causes countries can enter each other's markets with less effort. Imposing trade restrictions such as tariff and non-tariff trade barriers in a particular industry help to encourage domestic manufacturing of the industry, but decrease the volume and range of traded pharmaceutical products [bib_ref] Quantitative Analysis on the Impact of China-New Zealand FTA on Both Sides'..., Tan [/bib_ref]. Tariff rate is used in this study to measure the trade barriers for pharmaceutical products. It is assumed that TR i is negatively associated with Iran's intra-industry trade in pharmaceuticals.
In the next step of the analysis, the log-linear and log-log models are estimated and tested using diagnostic statistics to find a suitable model in order to investigate the impact of six independent variables on the Iran's IIT in pharmaceuticals.
LIIT iw = α 0 +α 1 PDIF i +α 2 ES i +α 3 MS i +α 4 DD i +α 5 KL i +α 6 TR i +u i
The logarithmic conversion of the Grubel-Lloyd index is calculated using the following function:
[formula] LIIT iw = log(6) [/formula]
It is expected that α 0 , α 1 , α 2 , α 3 , α 4 and α 5 will be positive, and α 6 will be negative.
The data used in the estimation of Iran's IIT in pharmaceuticals with the rest of the world were obtained from Iran's Ministry of Industry, Mine and Trade, Iran's pharmaceutical Statistics, the Islamic Republic of Iran's Customs Administration, and World Bank and International Trade Center. The seasonal time series for the period 2001-2012 were processed and coded according to a 4-digit level based on the International Classification's Harmonized System (3001 to 3006). Data were available in annual format, for increasing the number of observations we converted them to seasonal format. Data consisted of 48 observations for each variable. Also, the analysis was conducted on industry level, not at firm level.
# Results
The results presented in [fig_ref] Table 1: Comparison of Grubel-Lloyd index of intra-industry trade in pharmaceuticals for Iran and... [/fig_ref] show that Iran's intra-industry trade in pharmaceuticals with the rest of the world has increased from 5.97 in 2001 to 19.85 in 2012. As a result, the composition of Iran trade in pharmaceuticals has changed from inter-industry trade to intra-industry trade. Increasing the number of produced pharmaceuticals and improving product differentiation in this sector appear to be important reasons of the increase in the extent of Iran's intra-industry trade in pharmaceuticals. As a result, the rate of export growth has increased in the industry over the period. However, the level of Iran's intra industry trade in pharmaceuticals relative to other major exporting countries was small. Based on results of table 1, Italy, United Kingdom, Belgium, France, Germany and United States had high intra-industry trade indexes and were about net exporters of pharmaceuticals. On the contrary, the intra-industry trade index was partly low for Switzerland. Switzerland has the maximum revealed comparative advantage than all pharmaceutical exporting countries; therefore, Switzerland's IIT with the rest of the world was low since the pharmaceutical exports were much greater than its imports.
The results of diagnostic tests showed that for the model of Iran's intra-industry trade in pharmaceuticals with the rest of the world, the log-linear form was appropriate. Furthermore, Augmented Dickey-Fuller (ADF) test were used to test for the presence of unit root in each series of data. Due to non stationary of variables, we had to test for cointegration. The Engle-Granger test confirmed that the Long run relationships between the variables.
The estimated coefficients of the function for Iran's intra-industry trade in pharmaceuticals with the rest of the world along with the expected signs of the coefficients of the explanatory variables are represented in [fig_ref] Table 2: Iran's intra-industry trade in pharmaceuticals with the rest of the world [/fig_ref]. As shown in [fig_ref] Table 1: Comparison of Grubel-Lloyd index of intra-industry trade in pharmaceuticals for Iran and... [/fig_ref] , economies of scale (ES i ), market structure (MS i ), degree of economic development (DD i ) and trade barriers (TR i ) had statistically significant impacts on Iran's intra-industry trade in pharmaceuticals. The coefficients of product differentiation (PDIF i ) and technological advancement (KL i ) didn't have the expected signs; however, technological advancement variable unlike product differentiation variable was statistically significant.
As expected, economies of scale in Iran's pharmaceutical industry had a significantly positive effect on the extent of intra-industry trade. Market structure was positively related to Iran's intra-industry trade in pharmaceuticals with the rest of the world, which means an increase in the number of pharmaceutical companies will result in a higher extent of intra-industry trade. The coefficient of DD i was positive and significant at the 10 percent level. These results confirm that the degree of economic development has a positive influence on intra-industry trade. Therefore, an increase in income will cause a large demand for pharmaceutical products. The coefficient of TR i was negative and significant, indicating that tariff trade barriers have a negative impact on the extent of Iran's intra-industry trade in pharmaceuticals.
Contrary to expectations, the effect of product differentiation (PDIF i ) on Iran's intra-industry trade in pharmaceuticals was negative. Applying the ratio of R&D expenditures to value added as a criterion for product differentiation may have a negative impact on IIT in countries with a high technological advancement in that industry. Thus, Iran may have a partial technological strength in pharmaceutical production which causes this country to participate in low extent of intra-industry trade in pharmaceuticals than other exporting countries. This result was consistent with the finding of Karn.
During the study period, the ratio of capital-labor in Iran relative to the level of intra-industry trade in pharmaceuticals increased gradually. However, the level of technological advancement (KL i ) which was calculated by the ratio of total capital to the labor force had a significantly negative influence on Iran's IIT in pharmaceuticals with the rest of the world. Therefore, due to the estimated coefficient for KL i was negative, the impact of capital-labor ratio on Iran's IIT in pharmaceuticals wasn't supported by the model.
So, a one percent increase in the variables of product differentiation, economies of scale, market structure, degree of economic development, technological advancement and trade barriers will result in -9.27, 0.0086, 0.018, 0.00017, -0.17 and -0.0089 percent rise in Iran's intra-industry trade with the rest of the world, respectively. In this paper, when product differentiation and technological advancement rise, the extent of intra-industry trade will decline that are not consistent with theoretical expectations.
So far, one single study has been carried out to examine intra-industry trade in pharmaceuticals which has been performed by Karn in Australia. He has studied the models and factors affecting Australia's international trade in pharmaceutical sector. His research's results are largely similar to our research. According to Karn's findings, the variables of economies of scale, market structure and the degree of economic development had a significantly positive impact on Australia's IIT in pharmaceuticals, but trade barriers had a significantly negative influence.
# Discussion
Considering extensive literature review, we found 6 factors affecting the IIT in pharmaceuticals with the rest of the world. Overall, three variables affected IIT in pharmaceuticals in the same way. One was economies of scale, which had a significant and positive impact on IIT. Another factor was the influence of market structure. IIT was positively affected by market structure. The last factor was the degree of economic development in terms of per capita GNP. IIT is more likely to take place with large economies than with small ones. Tariff trade barriers had a significantly negative influence. These observations were in line with most theoretical predictions and empirical findings in the intra-industry trade literature. The estimated coefficients for product differentiation and the level of technological advancement of the country didn't have the expected positive signs, but the variable of the level of technological advancement was significant. Product differentiation had a negative impact on Iran's intra-industry trade and indicates that Iran may have technological intensity in pharmaceutical production leading to the country's engagement in less intra-industry trade in pharmaceuticals than other major exporting countries. Therefore, product differentiation is not necessary to increase IIT in every case, but it depends on the situation in which the index is applied. Nevertheless the steady increase in the capital-labor ratio in Iran relative to the extent of intra-industry trade in pharmaceuticals, the influence of the ratio of total capital to the labor force on Iran's intra-industry trade in pharmaceuticals was negative during the study period.
Also, Iran's IIT in pharmaceuticals have shown an increasing trend during the period 2001 to 2012. This growth may be due to an increase in the number of produced pharmaceuticals and product differentiation in this sector. The great intra-industry trade indexes for industrial countries seem to be associated with customs unions, high levels of income, common borders, trade liberalization and attainment to larger economies of scale in pharmaceutical production. Low levels of Iran's Intra-industry trade indicate that Iran is to some extent a net importer of pharmaceuticals.
Due to the low extent of Iran's intra-industry trade in pharmaceuticals, it seems that there is a considerable potential for developing and promoting intra-industry trade and exports in pharmaceutical industry. However, preparation for integration into the global economy in commodity groups having intra-industry trade is relatively high. Therefore, it seems that in order to get more prepared for integration into the global economy, Iran should pay special attention to promoting intra-industry trade in pharmaceuticals besides protecting the existing comparative advantages.
In order to increase Iran's IIT in pharmaceuticals, the most important recommendations are as follows:
1). New sources particularly product differentiation and economies of scale are important in establishing a sustainable advantage in pharmaceutical industry. Accordingly improving product differentiation, investing in skilled manpower, creating and extending research and development departments (R&D), and diversifying exports are essential. In addition, improving the level of technology, reducing costs and taking advantage of economies of scale are significant and can be taken into consideration.
2). Country-specific characteristics, such as foreign direct investment and active participation in economic integrations lead to the promotion of intra-industry trade in pharmaceutical industry. Hence, paying serious attention to these factors can result in an increase in pharmaceutical's intra-industry trade.
3). Development and promotion of intra-industry trade reduce pharmaceutical industry's demand for support. Therefore, since development of intra-industry trade involves both exports and imports, the pressure from support-seeking parties is reduced and a transparent, strong and permanent strategy can be developed. 4). Due to the chronic shortage of foreign exchange resources in Iran, attracting more foreign investment in pharmaceutical industry is of utmost importance. 5). It is necessary to reduce dependence on imported pharmaceutical primary materials in order to cope with the sharp increase in imports and maintain trade balance. Since pharmaceutical imports are mostly in the form of pharmaceutical primary materials, one of the most important strategies for increasing the competitiveness potential of Iran's pharmaceutical sector is to reduce import and achieve independence. 6). Developing a transparent and appropriate strategy for constant export of pharmaceutical products is essential for preventing instability in export and market loss.
pharmaceutical market that are considered as a threat to peoples' health and life.
# Conclusions
In summary, it is concluded that compared with other major exporting countries, the extent of Iran's intra-industry trade in pharmaceuticals was low. However, Iran's intra-industry trade in pharmaceuticals with the rest of the world was affected by industry characteristics such as economies of scale, market structure and so on. Given the low level of intra-industry trade in pharmaceutical products, the pharmaceutical competitiveness and globalization rates of Iran are small. So, in order to get ready for joining the global economy, Iran should consider the development of IIT in pharmaceuticals. Therefore, paying attention to intra-industry trade could have a valuable role in helping pharmaceutical companies and policy makers to promote pharmaceutical trade. Moreover, this work partly helps to clarify the path of Iran's pharmaceutical trade policy.
# Ethical issues
Ethical issues have been completely considered by the authors.
[table] Table 1: Comparison of Grubel-Lloyd index of intra-industry trade in pharmaceuticals for Iran and other major exporting countries with the rest of the world, selected years during 2001-2012 [/table]
[table] Table 2: Iran's intra-industry trade in pharmaceuticals with the rest of the world [/table]
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Reconstruction of epidemic curves for pandemic influenza A (H1N1) 2009 at city and sub-city levels
To better describe the epidemiology of influenza at local level, the time course of pandemic influenza A (H1N1) 2009 in the city of Hong Kong was reconstructed from notification data after decomposition procedure and time series analysis. GIS (geographic information system) methodology was incorporated for assessing spatial variation. Between May and September 2009, a total of 24415 cases were successfully geocoded, out of 25473 (95.8%) reports in the original dataset. The reconstructed epidemic curve was characterized by a small initial peak, a nadir followed by rapid rise to the ultimate plateau. The full course of the epidemic had lasted for about 6 months. Despite the small geographic area of only 1000 Km 2 , distinctive spatial variation was observed in the configuration of the curves across 6 geographic regions. With the relatively uniform physical and climatic environment within Hong Kong, the temporo-spatial variability of influenza spread could only be explained by the heterogeneous population structure and mobility patterns. Our study illustrated how an epidemic curve could be reconstructed using regularly collected surveillance data, which would be useful in informing intervention at local levels.
The time course of an infectious disease epidemic is one important piece of information for understanding the dynamics of pathogen transmission. For a localized outbreak, for example, food poisoning, an epidemic curve is often conveniently drawn during case investigation. Describing the time course of a country-wide epidemic is more complex, which is not uncommonly complicated by reporting delay, discrepant access to diagnostics, varied public perception and the influence of accompanying health-seeking behaviours. In time of an emerging pandemic, these obstacles pose a great challenge to our society, when a timely construction of an epidemic curve is desirable. The spread of pandemic (H1N1) 2009 was a case in point. When the pandemic first hit the population, most people were non-immune to the novel virus, albeit the presence of partial immunity in some older people [bib_ref] Crossreactive antibody responses to the 2009 pandemic H1N1 influenza virus, Hancock [/bib_ref]. The relative lack of airborne transmission implies that the dissemination of the virus could be shaped largely by population structures, their networking pattern and human mobility [bib_ref] Lack of airborne transmission during outbreak of pandemic (H1N1) 2009 among tour..., Han [/bib_ref]. An epidemic curve, if constructed, should reflect these characteristics for supporting the design of effective public health control programs.
Because of the spatial variability of the population, it is hypothesized that the epidemic curves could vary significantly from place to place. In this study we set out to describe the time course of the H1N1 epidemic with a spatial context in Hong Kong, a South-Eastern Chinese territory of about 1000 Km 2 in area. Since the diagnosis of the first case on 1 May 2009, all laboratory confirmed cases of pandemic (H1N1) 2009 were reported to the Government. Through the Centre for Health Protection, an anonymised dataset was obtained for the study, which included the age, gender and residential building location of each confirmed case. The residential address was transformed to x and y coordinates in Hong Kong Grid 1980 projection system. Geographically, Hong Kong can be divided into 18 districts and 400 District Council Constituency Areas (DCCA), each of the latter having an average of 17000 population for electoral purpose. ArcGIS version 9.2 was used for spatial exploration while time series analysis was performed to track the time course of the epidemic. A filtering procedure was applied to decompose the series into trend, seasonal and residual components (STL -seasonal trend decomposition procedure based on Loess), implemented on R [bib_ref] STL: a seasonal-trend decomposition procedure based on Loess, Cleveland [/bib_ref]. Institutional approval for access to the data was obtained from HKSAR Department of Health, in compliance with the Personal Data (Privacy) Ordinance.
Individual consent was deemed unnecessary in the analysis of collected surveillance data which did not involve primary data collection.
Overall, a total of 24415 pandemic (H1N1) 2009 cases were successfully geocoded, out of 25473 (95.8%) reported between May and September 2009. The male- to-female ratio was 1.07:1. There was marked heterogeneity in the geographic spread of the reported cases, ranging from 6 cases to 272 cases per DCCA (figure 1). Evaluating at district level, the number of reported cases ranged from below 30 to > 50 per 100,000 populations.
In the absence of physical boundaries between geographic units, people are free to move within and across districts and DCCA in their daily activity. We redefined six geographic regions representing places separated by natural borders like mountains and water bodies, after exclusion of uninhabitable areas. The region boundaries, population size and demographic characteristics of pandemic influenza (H1N1) 2009 cases are given in [fig_ref] Figure 1: Spatial distribution of reported Pandemic influenza [/fig_ref] and [fig_ref] Table 1: Population characteristics by geographic region [/fig_ref]. The attack rate, expressed as the reported number per 100 resident population was similar across all regions, despite the difference in case density. The proportion of students (defined as people of age [bib_ref] Characterizing the initial diffusion pattern of pandemic (H1N1) 2009 using surveillance data, Lee [/bib_ref] [bib_ref] School closure and mitigation of pandemic (H1N1) 2009, Hong Kong, Cowling [/bib_ref] [bib_ref] Surveillance lessons from first-wave pandemic (H1N1), Baxter [/bib_ref] [bib_ref] The importance of school and social activities in the transmission of influenza..., Kar-Purkayastha [/bib_ref] [bib_ref] Influenza surveillance by age and target group, Foy [/bib_ref] [bib_ref] Syndromic surveillance: STL for modeling, visualizing, and monitoring disease counts, Hafen [/bib_ref] in the reported case was higher than adults (age 20-64), a pattern opposite to that in the general population [fig_ref] Table 2: Characteristics of Pandemic influenza A [/fig_ref]. An epidemic curve was drawn from the reported numbers in the original dataset [fig_ref] Figure 2: Construction of epidemic curves using the original data [/fig_ref]. In this study, the parameters and components of STL function (Y t = T t +S t +R t ) were: t as the time unit, from 1 to 153; Y t as the daily count of H1N1 cases on day t; T t as the trend component; S t as the seasonal component, using 7-day as the smoothing window to account for the weekly cycles adopted by laboratories in the testing and reporting of results (tests on 3-day and 14-day windows were performed yielding less satisfactory results); and R t as the residual component. The final epidemic curve was reconstructed from the trend component after seasonal decomposition and the exclusion of residuals, through a sequence of operations employing Loess smoother. The Loess regression ĝ(x) smoothed y given x along the scale of the independent variable. The trend smoothing was computed in R by Loess [bib_ref] STL: a seasonal-trend decomposition procedure based on Loess, Cleveland [/bib_ref]. The regression was locally
[formula] weighted by V x W X X x i i q ( ) ( ( ) ) = − [/formula]
where W was the weighted function, λ q (x) was the qth farthest distance of x i from x, for i = 1 to n, with q as positive integer. The resultant trend (figure 2 lower panel) is characterized by a small peak at around Day 55-60, followed by a nadir and then rapid rise to the ultimate peak on Day 135. By defining students as those between the age of 5 and 19, the trend was plotted again using data on students alone and non-students, with the former bearing remarkable similarity to the all case series (results not shown). There were also marked variations across the 6 geographic regions in amplitude and configuration (figure 3). The early peak could only be seen in Kowloon, and less remarkably on Hong Kong Island region. The peak was reached in all 6 regions at around the same time, though the magnitude and the interval between onset and peak varied. The temporal profiles of residuals, constituted by the remains of the original dataset after seasonal and trend decomposition, [bib_ref] STL: a seasonal-trend decomposition procedure based on Loess, Cleveland [/bib_ref] demonstrate that there were more spikes over time on Hong Kong Island and Kowloon Peninsula(figure 4). Our study illustrated how an epidemic curve of an influenza epidemic could be reconstructed using regularly collected surveillance data, after decomposition of the time series. STL was applied on the assumption that the seasonal components had been contributed by weekly cycles of laboratory workloads, and that residuals reflected local outbreaks especially in schools. The smoothed trend therefore depicts the time course of the pandemic over a five-month period. The full time course of the epidemic could not be drawn as reporting of individual cases of pandemic (H1N1) 2009 ceased to be mandated at the end of September 2009. Interestingly, though, the peak appeared to have been reached before the reporting mechanism ended. A review of the epidemic curve constructed from influenza-like illness (ILI) surveillance suggested that this first wave came to the trough as of the end of October. It is therefore quite likely that the full course of the epidemic has lasted for about 6 months.
Despite the small area of the territory of Hong Kong, spatial variation was observed in its initial diffusion [bib_ref] Characterizing the initial diffusion pattern of pandemic (H1N1) 2009 using surveillance data, Lee [/bib_ref]. This phenomenon was further characterized in this study by the demonstration of variability in: (a) the time point at which influenza cases were first introduced, (b) the time for a critical mass of cases to become cumulated, before the epidemic kicked off, and (c) the amplitude of the regional epidemics. Of note were the small peak and a subsequent nadir in the initial part of the time course, which have been attributed to mitigation introduced by the Government through school closure [bib_ref] School closure and mitigation of pandemic (H1N1) 2009, Hong Kong, Cowling [/bib_ref]. The initial small peak was however not seen in all geographic regions. This may be explained by the differential pattern of virus transmissions in each region in view of the variation of population structures, or that there were higher uptake of reported cases in some locations against the background of considerable public attention, when the news of an impending epidemic first broke out [bib_ref] Surveillance lessons from first-wave pandemic (H1N1), Baxter [/bib_ref]. On the other hand, the landscape of the epidemic curve thus constructed was contributed largely by infections in students, an observation made in other studies on pandemic (H1N1) as well as seasonal influenza [bib_ref] The importance of school and social activities in the transmission of influenza..., Kar-Purkayastha [/bib_ref] [bib_ref] Influenza surveillance by age and target group, Foy [/bib_ref]. Against the background of a relatively uniform physical and climatic environment within Hong Kong, the temporo-spatial variability of influenza spread could only be explained by the heterogeneous population structure and mobility patterns.
Our study carried some limitations. Firstly we assumed that case reporting had been consistently executed over time. In the first five months, all clinically suspicious cases presenting to government clinical services and designated clinics were tested for the virus, alongside referrals from the private sector. While reporting can never be complete, the large number of cases reported (over 20,000) and the single public health agency supervising influenza surveillance in Hong Kong should have offset any inconsistency, thereby enhancing the robustness of the analysis. Secondly, STL decomposition was a filtering procedure based on an algorithm which may not have incorporated all determinants of the influenza transmission dynamics. The validity of the methods for field study would need to be further evaluated. Todate, STL has been used in syndromic surveillance, but with a different public health objective of detecting of outbreaks in the community [bib_ref] Syndromic surveillance: STL for modeling, visualizing, and monitoring disease counts, Hafen [/bib_ref]. In the development of an effective public health response, timeliness of the analysis and the use of regularly collected data are often crucial. In this connection, the algorithm described in this study has allowed the time course of a new epidemic to be drawn without resorting to sophisticated modeling techniques or simulations. The spatial variation in the time course of the pandemic (H1N1) 2009 was an important observation which may be further explored in context of strategies of public health interventions.
[fig] Figure 1: Spatial distribution of reported Pandemic influenza (H1N1) 2009 cases in Hong Kong from May to September 2009, by district council constituency area (DCCA). Uninhabitable areas, including land elevated over 200 meters and water bodies, are hashed.Table 1displays the population characteristics and statistics on reported cases for the 6 geographic regions -Hong Kong Island, Kowloon, North West, Sai Kung, Shatin/Tai Po, South West, the boundaries of which are given in thick lines. [/fig]
[fig] Figure 2: Construction of epidemic curves using the original data (upper panel) and then by time series analysis after seasonal decomposition of time series by Loess (STL) and smoothing (lower panel). The procedure involved five steps of, firstly, detrending; secondly, cycle-subseries smoothing by loess; thirdly, low-pass filtering by moving average; fourthly, detrending by subtracting seasonal component; and finally, deseasonalizing. [/fig]
[table] Table 1: Population characteristics by geographic region (2006 by-census data) in Hong Kong [/table]
[table] Table 2: Characteristics of Pandemic influenza A (H1N1) cases by geographic region (May-September 2009) in Hong Kong [/table]
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Pregnancy loss and role of infant HIV status on perinatal mortality among HIV-infected women
Background: HIV-infected women, particularly those with advanced disease, may have higher rates of pregnancy loss (miscarriage and stillbirth) and neonatal mortality than uninfected women. Here we examine risk factors for these adverse pregnancy outcomes in a cohort of HIV-infected women in Zambia considering the impact of infant HIV status. Methods: A total of 1229 HIV-infected pregnant women were enrolled (2001)(2002)(2003)(2004) in Lusaka, Zambia and followed to pregnancy outcome. Live-born infants were tested for HIV by PCR at birth, 1 week and 5 weeks. Obstetric and neonatal data were collected after delivery and the rates of neonatal (<28 days) and early mortality (<70 days) were described using Kaplan-Meier methods.Results: The ratio of miscarriage and stillbirth per 100 live-births were 3.1 and 2.6, respectively. Higher maternal plasma viral load (adjusted odds ratio [AOR] for each log10 increase in HIV RNA copies/ml = 1.90; 95% confidence interval [CI] 1.10-3.27) and being symptomatic were associated with an increased risk of stillbirth (AOR = 3.19; 95% CI 1.46-6.97), and decreasing maternal CD4 count by 100 cells/mm 3 with an increased risk of miscarriage (OR = 1.25; 95% CI 1.02-1.54). The neonatal mortality rate was 4.3 per 100 increasing to 6.3 by 70 days. Intrauterine HIV infection was not associated with neonatal morality but became associated with mortality through 70 days (adjusted hazard ratio = 2.76; 95% CI 1.25-6.08). Low birth weight and cessation of breastfeeding were significant risk factors for both neonatal and early mortality independent of infant HIV infection.Conclusions: More advanced maternal HIV disease was associated with adverse pregnancy outcomes. Excess neonatal mortality in HIV-infected women was not primarily explained by infant HIV infection but was strongly associated with low birth weight and prematurity. Intrauterine HIV infection contributed to mortality as early as 70 days of infant age. Interventions to improve pregnancy outcomes for HIV-infected women are needed to complement necessary therapeutic and prophylactic antiretroviral interventions.
# Background
In 2009, an estimated 1.4 million pregnant women were living with HIV in low-and middle-income countries. About 91% of those women in need of antiretroviral drugs for preventing mother-to-child transmission were in sub-Saharan Africa and 54% of women are estimated to have received the prophylactic drugs with recent scale-up. Besides the risk of HIV transmission, maternal HIV infection is associated with increased risk of adverse pregnancy outcomes. For example, a metaanalysis of 31 studies showed almost a four-fold increased risk of stillbirth among HIV-infected compared to uninfected women [bib_ref] The association between maternal HIV infection and perinatal outcome: a systematic review..., Brocklehurst [/bib_ref] , although some studies have not observed this [bib_ref] Impact of maternal HIV infection on obstetrical and early neonatal outcome, Braddick [/bib_ref].
Several studies in sub-Saharan African countries have also shown that infants born to HIV-infected women have a significantly increased risk of low birth weight (LBW) [bib_ref] Impact of maternal HIV infection on obstetrical and early neonatal outcome, Braddick [/bib_ref] [bib_ref] Study Group HIV: Low birthweight in infants born to African HIV-infected women:..., Castetbon [/bib_ref] [bib_ref] Maternal human immunodeficiency virus-1 infection and pregnancy outcome, Temmerman [/bib_ref] [bib_ref] Perinatal transmission of the human immunodeficiency virus type 1 to infants of..., Ryder [/bib_ref] , preterm birth [bib_ref] Study Group HIV: Low birthweight in infants born to African HIV-infected women:..., Castetbon [/bib_ref] [bib_ref] Maternal human immunodeficiency virus-1 infection and pregnancy outcome, Temmerman [/bib_ref] , neonatal mortality [bib_ref] Perinatal transmission of the human immunodeficiency virus type 1 to infants of..., Ryder [/bib_ref] and infant mortality [bib_ref] Perinatal transmission of the human immunodeficiency virus type 1 to infants of..., Ryder [/bib_ref] [bib_ref] The effect of human immunodeficiency virus infection on birth weight, and infant..., Taha [/bib_ref] [bib_ref] Timing of mother-to-child transmission of HIV-1 and infant mortality in the first..., Zijenah [/bib_ref]. Generally, markers of more advanced maternal disease, such as low CD4 cell counts and high plasma viral loads, are associated with these endpoints [bib_ref] Timing of mother-to-child transmission of HIV-1 and infant mortality in the first..., Zijenah [/bib_ref] [bib_ref] Effect of maternal CD4 + cell count, acquired immunodeficiency syndrome, and viral..., Lambert [/bib_ref] [bib_ref] Perinatal AIDS Collaborative Transmission Study (PACTS) Group: Maternal health factors and early..., Abrams [/bib_ref]. However, since these markers are also strongly related to HIV transmission [bib_ref] The effect of maternal viral load on the risk of perinatal transmission..., Thea [/bib_ref] [bib_ref] Maternal levels of plasma human immunodeficiency virus type 1 RNA and the..., Garcia [/bib_ref] [bib_ref] Risk factors for perinatal transmission of human immunodeficiency virus type 1 in..., Mofenson [/bib_ref] , it is not clear whether infant HIV infection is a cause or a consequence of these adverse events. One study that separated HIV infections by their timing found that preterm birth was associated with intrapartum but not with intrauterine infection, suggesting prematurity as a risk factor for intrapartum infection rather than a cause of preterm delivery [bib_ref] Distinct risk factors for intrauterine and intrapartum human immunodeficiency virus transmission and..., Kuhn [/bib_ref]. In some studies, intrauterine-infected children had smaller weight-for-gestation than expected, suggesting some growth consequences of early intrauterine HIV infection [bib_ref] Determinants of low birth weight among HIV-infected pregnant women in Tanzania, Dreyfuss [/bib_ref] [bib_ref] New York City Perinatal HIVTCSG: Timing of maternal-infant HIV transmission: associations between..., Kuhn [/bib_ref].
HIV-infected infants have a substantially higher risk of infant mortality compared to uninfected but exposed infants [bib_ref] Mortality of infected and uninfected infants born to HIV-infected mothers in Africa:..., Newell [/bib_ref] [bib_ref] The Zvitambo Study Group: Child mortality according to maternal and infant HIV..., Marinda [/bib_ref]. In addition, infants infected intrauterine or intrapartum have a worse prognosis than those infected through breastfeeding [bib_ref] The Zvitambo Study Group: Child mortality according to maternal and infant HIV..., Marinda [/bib_ref] [bib_ref] Reduced mortality associated with breastfeeding-acquired HIV infection and breast-feeding among HIV-infected children..., Fox [/bib_ref] [bib_ref] Association between low birth weight and infant mortality in children born to..., Wei [/bib_ref]. But it is unclear whether perinatally-acquired HIV infection will influence infant survival within the first months after birth. Since LBW and preterm birth are the major risk factors for neonatal and early infant mortality in all populations regardless of HIV status, separating out independent effects of these inter-related perinatal variables is complex and requires large sample sizes [bib_ref] The contribution of low birth weight to infant mortality and childhood morbidity, Mccormick [/bib_ref] [bib_ref] Preventing Preterm Birth and Neonatal Mortality: exploring the Epidemiology, Causes, and Interventions, Simmons [/bib_ref]. One large study in Tanzania found that among infants born to HIV-infected mothers, LBW, not infant HIV infection, was the strongest risk factor for neonatal mortality [bib_ref] Association between low birth weight and infant mortality in children born to..., Wei [/bib_ref].
Here we investigate risk factors for miscarriage, stillbirth, neonatal mortality and early mortality among a cohort of pregnant HIV-infected women followed as part of a study in Lusaka, Zambia. We aim to identify independent contributions of advanced maternal disease and infant HIV status to adverse pregnancy and neonatal outcomes and focus on whether the timing of infant HIV infection influences the risk of these adverse outcomes.
# Methods
## Study design
We report a secondary analysis of data collected as part of the Zambia Exclusive Breastfeeding Study (ZEBS), a randomized clinical trial to examine whether exclusive breastfeeding to 4 months, followed by abrupt weaning, could reduce postnatal HIV transmission and child mortality. The study protocol and primary results have been reported elsewhere [bib_ref] Issues in the design of a clinical trial with a behavioral intervention-the..., Thea [/bib_ref] [bib_ref] Effects of early, abrupt weaning on HIV-free survival of children in Zambia, Kuhn [/bib_ref] [bib_ref] Elevations in mortality associated with weaning persist into the second year of..., Kuhn [/bib_ref]. Briefly, a total of 1435 HIV-positive pregnant women were recruited at two antenatal care clinics in Lusaka between May 2001 and September 2004 if they intended to breastfeed, accepted to take single-dose nevirapine to prevent perinatal HIV transmission and consented to be assigned to either of the study and control groups. Antiretroviral therapy (ART) only became available in the public sector as the study was nearing completion. Written informed consent was obtained from all study participants. The study was approved by human subjects committees at all collaborating institutions.
## Study procedures
Socioeconomic and clinical data were collected at enrollment. Maternal blood samples were drawn at enrollment to test for CD4 cell counts (FACSCount; BD Immunocytometry Systems), hemoglobin level (Hemocue system), and plasma viral load (Roche Amplicor 1.5; Roche). All women with positive results of routine syphilis screening test (RPR) were treated with penicillin. Two subsequent antenatal visits were scheduled before delivery. Data on the circumstances of miscarriages and stillbirths were collected from medical records if available or from interview with the mothers. Gestational week at delivery was calculated from the estimated gestational week at enrollment, which was based on clinical examination (fundal height) and self-report of last menstrual period (LMP). In cases of discrepant gestational age estimates, the LMP date was used. Heel stick blood samples of infants were obtained at clinic visits scheduled at birth, 1 week and 5 weeks. DNA was extracted from each infant's blood sample and tested for HIV by real-time polymerase chain reaction (PCR). All positive test results were confirmed by testing at least two samples. To rule out false negative test results due to an inadequate sample, amplification of the beta-globin gene was performed to ensure adequate cell numbers. Home visits were scheduled at 4 days after birth and 2, 3 and 7 weeks post-partum. Study participants who did not return for the scheduled antenatal or clinic visits were tracked and followed by home-visit teams.
## Study variables
The primary outcomes of interest were miscarriage, stillbirth, neonatal mortality (infant's death <28 days) and early mortality (infant's death <70 days). Miscarriage was defined as spontaneous abortion of pregnancy occurring before 24 gestational weeks, and stillbirth as fetal death occurring at 24 gestational weeks or later. Low birth weight (LBW) was defined as birth weight <2500 g, and preterm birth as having <34 weeks of gestation. This cut-off was selected since systematic under-estimation of gestational age by 2-3 weeks was indicated by the birth weight distribution. 30 infants did not have birth weight data mainly due to delivery at home. Their birth weights were predicted based on their weights measured at 1 week.
Maternal body mass index (BMI) at 1 month postpartum was used as a predictor since women were enrolled at different gestational weeks and often quite late in pregnancy. 123 women who missed the one month visit were included into models as a separate group. Breastfeeding was defined as having stopped only if cessation occurred before the illness preceding infant's death or loss to follow up. Stopping breastfeeding was considered as a fixed-covariate and a time-dependent variable. After November 2003, cotrimoxazole prophylaxis was introduced for women with CD4 cell counts <200 cells/mm 3 and the effects of cotrimoxazole were reported elsewhere [bib_ref] Reduction in preterm delivery and neonatal mortality after the introduction of antenatal..., Walter [/bib_ref]. If women experienced weight loss, >30 days of diarrhea, fever or cough in the six months prior to enrollment, or had history of thrush or tuberculosis, women were considered as being symptomatic according to World Health Organization criteria. Further, each symptom was individually examined.
The timing of HIV transmission was stratified into three groups: 1) intrauterine transmission (IU) 2) intrapartum/early post-partum transmission (IP/ePP) and 3) no infection within 42 days after birth (NI). IU transmission was presumed if PCR results were positive within 3 days of birth. IP/ePP transmission was presumed if the PCR results were positive between 4 and 42 days after birth. If PCR results were negative at least for 42 days after birth, then the infants were presumed not to be infected within 42 days (NI). Mortality among the three groups with different timing of HIV transmission was compared. The cut-off point of early mortality was chosen at 70 days to measure the effect of IU and IP infection over the 6 weeks period after the neonatal period. Those with no PCR results were considered to be of unknown HIV status.
# Statistical analysis
Mantel-Haenszel relative risk (RR) of each factor on each outcome was examined and reported with 95% Confidence Intervals (CIs). For adjusted analysis, multivariable logistic regression was fitted. Variables with 'pvalue < .10 in univariable analysis were included and were retained if significant (p-value <0.05) and their odds ratios (ORs) were reported with 95% CIs. Kaplan-Meier methods were used to calculate mortality rates for IU, IP/ePP and NI infants and Cox Proportional Hazards models to examine risk factors of neonatal mortality and early mortality. Unadjusted and adjusted hazard ratios (HRs) were reported with 95% CIs. Continuous and categorical variables were compared across groups by two sided-Wilcoxon tests or t-tests and χ 2 statistics, respectively. All statistical analyses were performed by SAS (version 9.2).
# Sensitivity analysis
Sensitivity analysis was performed to examine whether infants with unknown HIV status biased the results. First, we assumed that all infants with unknown status are IU, IP or NI. Next we estimated the probability of IU or IP transmission in the infants of unknown HIV status among those who died or who were lost to follow up separately based on a logistic regression model predicting transmission from maternal CD4 cell counts and plasma viral load. Finally, we calculated the expected neonatal and early mortality rates that would have occurred had these transmission rates occurred in each group of IU, IP and NI infants.
# Results
## Cohort description
Of 1435 HIV-positive women in the study, 11 women were not pregnant, 191 withdrew or were lost to follow up and four women died before delivery. Of 1229 women, 3.4% were enrolled during the first trimester and 62.7% during the second (median 26 weeks; IQR 20-31). There were 36 miscarriages and 30 stillbirths. Thus, 1163 women gave birth to 1185 live-born infants (26 non-singleton births including 21 pairs of twins both born alive). For 53 women, live-births were known to have occurred but no delivery or follow-up data were available. Compared to all 1163 live-born infants, the ratio of miscarriage and stillbirth per 100 live-births were 3.1 (95% CI 2.2-4.3) and 2.6 (95% CI 1.8-3.7), respectively. Among 1132 live-born infants with delivery records from 1110 mothers, 14.3% had LBW, and 16.3% were preterm. The probabilities of neonatal (<28 days) and early (<70 days) mortality were 0.043 (n = 47, 95% CI 0.032-0.057) and 0.063 (n = 67, 95% CI 0.050-0.079), respectively [fig_ref] Table 1: Frequencies of adverse pregnancy outcomes among 1229 HIV-infected pregnant women recruited in... [/fig_ref]. No woman with miscarriage or stillbirth had started ART and only 6 women started ART during pregnancy and 3 within 70 days post-partum.
## Risk factors for miscarriage and stillbirth
Risk factors for miscarriage or stillbirth were examined [fig_ref] Table 2: Risk factors for miscarriage and stillbirth among a cohort of 1229 HIV-infected... [/fig_ref]. CD4 cell counts <350 cells/mm 3 and history of miscarriage were associated with an increased risk of miscarriage. For stillbirth, plasma viral load ≥50,000/ml and symptomatic HIV disease were significant risk factors as well as no previous pregnancies. In multivariable analysis, only CD4 cell counts <350 cells/mm 3 was significantly associated with miscarriage. The odds ratio for decreasing CD4 cell counts by 100 cells/mm 3 was 1.25 (95% CI 1.02-1.54, p = 0.03). For stillbirth, plasma viral load (adjusted OR [AOR] for each log10 increase in HIV RNA copies/ml = 1.90; 95% CI 1.10-3.27), being symptomatic (AOR = 3.19; 95% CI 1.46-6.97) and no previous pregnancy (AOR = 2.71; 95% CI 1.16-6.31) remained as significant risk factors.
## Risk factors for lbw and preterm birth
Risk factors for LBW and preterm birth are shown in [fig_ref] Table 3: Risk factors for low birth weight [/fig_ref]. Higher plasma viral load, BMI <18.5 kg/m 2 and swollen glands in more than one place were associated with both LBW and premature birth. In addition, women with CD4 cell counts <350 cells/mm 3 had an increased risk of having LBW infants (RR = 1.57; 95% CI 1.16-2.12) compared to women with higher CD4 cell counts. Infants born to mothers with hemoglobin <10 g/dl, clinical symptoms or history of tuberculosis were more likely to be LBW. Less formal education was associated with preterm birth and women aged >30 years had a lower risk of preterm birth than women aged 20-30 years.
In multivariable analysis, maternal plasma viral load (AOR for each log10 increase = 1.30; 95% CI 1.03-1.66), BMI <18.5 kg/m 2 (AOR = 2.35; 95% CI 1.49-3.70), hemoglobin <10g/dl (AOR = 1.50; 95% CI 1.04-2.16), a history of swollen glands in more than one place (AOR = 1.64; 95% CI 1.02-2.63) or tuberculosis (AOR = 1.85; 95% CI 1.11-3.06) were each independently associated with a higher risk of LBW. For preterm birth, BMI <18.5 kg/m 2 was the strongest risk factor (AOR = 1.96; 95% CI 1.27-3.04). History of swollen glands (AOR = 1.77; 95% CI 1.14-2.73) and less formal education (AOR = 1.42; 95% CI 1.00-2.02) were independently associated with preterm birth. Maternal age >30 years was still associated with reduced risk of preterm birth (AOR = 0.62; 95% CI 0.38-1.00).
## Risk factors for neonatal and early mortality
Risk factors for neonatal and early mortality are shown in [fig_ref] Table 4: Risk factors for neonatal mortality [/fig_ref]. Preterm birth, LBW, APGAR scores and cesarean delivery were strongly associated with an increased risk of both neonatal and early mortality in univariable analysis. History of tuberculosis and >30 days of diarrhea were significant risk factors for both neonatal and early mortality. Relative risks of these factors were higher for neonatal mortality than for early mortality. Maternal health factors, namely CD4 cell counts <350 cells/mm 3 , hemoglobin <10 g/dl, higher plasma viral load, and BMI <18.5 kg/m 2 were significantly associated with an increased risk of early mortality, but not with neonatal mortality.
Maternal death <70 days was associated with an increased risk of both neonatal and early mortality but many of these deaths occurred after infant death. Restricting only to maternal deaths occurring before infant death (18/1132; 1%), the association with early mortality was attenuated. Stopping breastfeeding <70 days was a significant risk factor for neonatal and early mortality. When fitted as a time-dependent variable, the association was further strengthened for both neonatal mortality (HR = 15.02; 95% CI 5.20-43.43) and early mortality (HR = 15.37; 95% CI 7.39-31.97). Infants born to mothers aged <20 years had a two-fold increased risk of neonatal and early mortality and having more than one child aged 5-16 years living in the household was associated with a lower risk of early mortality.
In multivariable analysis, LBW, stopping breastfeeding and young maternal age were significantly associated with both neonatal and early mortality. Preterm birth was only associated with neonatal mortality and low maternal CD4 cell counts (<350 cells/mm 3 ) only with early mortality [fig_ref] Table 5: Multivariate analysis of risk factors for neonatal and early mortality a [/fig_ref] , Model 1).
## Effects of infant hiv status
Among the 1132 infants, 50 infants had no HIV test results. Of 1082 infants with a known HIV status, 64 infants (5.9%) were IU infected. Another 64 infants (5.9%) were IP/ePP infected including 10 infants with indeterminate HIV status at birth but tested HIVpositive by 42 days. 863 infants were followed up and not infected at least for 42 days. 69 infants were lost to follow up but confirmed HIV-negative prior to 42 days and another 22 infants were HIV-negative at birth or [fig_ref] Table 5: Multivariate analysis of risk factors for neonatal and early mortality a [/fig_ref] shows the associations between IU and IP/ePP transmission and neonatal and early mortality in multivariable model. IU infection remained significantly associated with early mortality (AHR = 2.76; 95% CI 1.25-6.08) after adjustment for LBW, preterm birth, stopping breastfeeding, and having more than a child aged 5-16 years. IP/ePP infection was not significantly associated with early mortality and cannot be directly examined in relation to neonatal mortality since survival to a subsequent test was a component of the definition of intrapartum infection. Major causes of neonatal and early mortality were similar among the three groups; pneumonia, acute diarrhea and septicemia. When we examined the mortality between 28 and 70 days, the risk of the mortality among IU infants was and stopping breastfeeding (AHR = 9.52; 95% CI 3.65-24.85) were significantly associated with early mortality as seen in whole population.
# Sensitivity analysis
The 50 infants with unknown HIV status had lower birth weights and higher mortality rates than IU, IP and NI infants. 23 infants (46%) died by 28 days and 24 infants (48%) by 70 days. To determine the extent of bias, we performed a sensitivity analysis including these infants under different assumptions [fig_ref] Table 6: Sensitivity analyses for the 50 infants with unknown HIV status a Assume... [/fig_ref].
The predicted probability of HIV transmission was 6.8% for IU and 6.9% for IP in the 24 infants who died before 70 days and 4.4% for IU and 4.0% for IP in the 26 infants lost to follow up. Assuming these transmission rates among the infants with unknown HIV status, we calculated neonatal mortality rates of 7.3% in IU, 3.9% in IP and 4.1% in NI groups. Early mortality rates were 15.6% in IU, 7.1% in IP and 5.5% in NI groups among the 1132 infants. After this imputation, the adjusted HRs of neonatal mortality and early mortality among IU transmitted infants were 1.66 (95% CI 0.65-4.29) and 2.09 (95% CI 1.05-4.17) compared to NI infants, respectively (Model 4). This is similar to the results (24) Food run out at least 1 day in last month excluding the infants of unknown HIV status suggesting that the original approach was reasonable.
# Discussion
This study documents that women with advanced HIV disease are not only more likely to transmit HIV but also have an increased risk of miscarriage and stillbirth and infants born to these mothers have an increased risk of neonatal and early mortality. High rates of neonatal and early mortality are primarily attributable to low birth weight and preterm birth, both also increased among women with advanced disease. It is only after the neonatal period that the infant's HIV status begins to make a significant contribution to excess mortality. In this study, the risks of miscarriage and stillbirth were 31 and 26 per 1000 live-born births, respectively. From another cohort study in Lusaka, Zambia, the estimated rate of stillbirth was 21 per 1000 live-born births where 75.2% of the participating women were HIVuninfected [bib_ref] Determinants of stillbirth in Zambia, Stringer [/bib_ref]. We found that among HIV-infected women, miscarriage was associated with CD4 cell counts <350 cells/mm 3 . This result is similar to other studies [bib_ref] Pregnancy outcomes in HIV-infected and uninfected women in rural and urban South..., Rollins [/bib_ref] [bib_ref] Impact of maternal HIV-1 infection on perinatal outcome, Kumar [/bib_ref]. In a South African cohort study, women with CD4 cell counts <250 cells/mm 3 had a two-fold increased risk of the adverse outcome (antenatal heath, miscarriage or stillbirth) compared to those with CD4 cell counts >500 cells/mm 3 [bib_ref] Pregnancy outcomes in HIV-infected and uninfected women in rural and urban South..., Rollins [/bib_ref]. Another study in India reported that symptomatic women had a significantly increased rate of miscarriage, compared to asymptomatic women [bib_ref] Impact of maternal HIV-1 infection on perinatal outcome, Kumar [/bib_ref].
In our study, high plasma viral load was significantly associated with stillbirth among HIV-infected women. Many studies have reported increased risk of stillbirth in HIV-infected women compared to HIV-negative women [bib_ref] The association between maternal HIV infection and perinatal outcome: a systematic review..., Brocklehurst [/bib_ref] , but only two studies have examined risk factors associated with stillbirth among HIV-infected pregnant women [bib_ref] Predictors of stillbirth among HIV-infected Tanzanian women, Kupka [/bib_ref]. A study in Tanzania found that elevated CD3 count, but not other markers such as CD4 or CD8 counts, was associated with stillbirth [bib_ref] Predictors of stillbirth among HIV-infected Tanzanian women, Kupka [/bib_ref]. In the cohort conducted in four cities in sub-Saharan Africa, the risk of stillbirth was correlated with decreasing CD4 countbut not with plasma viral load categorized into ≥100,000 copies/ml or less. In our analysis, the mean baseline plasma viral load of mothers who delivered stillbirths was significantly higher (median value 76,560 vs. 39,783 copies/ml, p = 0.01) thus fitting it as a continuous variable (log scale) seemed to be more accurate. Furthermore, symptomatic women had a more than a three-fold increased risk of stillbirth compared to asymptomatic women, suggesting that maternal disease progression may play an important role in perinatal outcomes.
Some previous studies have tried to document HIV status of pregnancy losses [bib_ref] Maternofetal transmission of AIDS: frequency of human immunodeficiency virus type 1 nucleic..., Soeiro [/bib_ref] [bib_ref] Excess intrauterine fetal demise associated with maternal human immunodeficiency virus infection, Langston [/bib_ref] [bib_ref] Frequency of early in utero HIV-1 infection: a blind DNA polymerase chain..., Brossard [/bib_ref]. One study which did so by in situ hybridization showed that 60% of the spontaneous fetal losses (6/10) were associated with HIV transmission to the fetus [bib_ref] Excess intrauterine fetal demise associated with maternal human immunodeficiency virus infection, Langston [/bib_ref]. It is possible that advanced maternal disease might have increased pregnancy losses via intrauterine transmission [bib_ref] Risk factors for in utero and intrapartum transmission of HIV, Magder [/bib_ref] [bib_ref] Risk factors for in utero or intrapartum mother-to-child transmission of human immunodeficiency..., Jourdain [/bib_ref] but we could not directly examine such mechanism. Since ART only became available towards the end of the study, 52 out of 66 women (79%) who had miscarriages or stillbirths should have received ART under the current WHO guidelines but did not. Although certain ART regimens may increase the risk of adverse pregnancy outcomes [bib_ref] Association of HIV infection with spontaneous and iatrogenic preterm delivery: effect of..., Lopez [/bib_ref] [bib_ref] Antiretroviral therapy during pregnancy and the risk of an adverse outcome, Tuomala [/bib_ref] , it also reduces the risk of perinatal transmission and improves maternal health [bib_ref] Antiretroviral regimens in pregnancy and breast-feeding in Botswana, Shapiro [/bib_ref]. Timely ART for HIV-infected pregnant women may result in a net improvement in these perinatal outcomes.
Our data indicate that HIV infection acquired during pregnancy begins to contribute to excess mortality as early as 70 days but mostly only after the neonatal period, independent of LBW and early cessation of breastfeeding. After the neonatal period, IU-infected infants had more than a six-fold increased risk of mortality compared to NI infants through 70 days. Prior studies have not as precisely considered the timing of early mortality of infected infants [bib_ref] The Zvitambo Study Group: Child mortality according to maternal and infant HIV..., Marinda [/bib_ref] [bib_ref] Reduced mortality associated with breastfeeding-acquired HIV infection and breast-feeding among HIV-infected children..., Fox [/bib_ref] nor separated IU transmission from IP/ePP transmission [bib_ref] Association between low birth weight and infant mortality in children born to..., Wei [/bib_ref]. Some studies have found IU-infected infants to have onset of symptoms in first few months of life and faster disease progression than those infected at delivery or later [bib_ref] Distinct risk factors for intrauterine and intrapartum human immunodeficiency virus transmission and..., Kuhn [/bib_ref] [bib_ref] High frequency of rapid immunological progression in African infants infected in the..., Mphatswe [/bib_ref] [bib_ref] Viral load and disease progression in infants infected with human immunodeficiency virus..., Shearer [/bib_ref]. Despite recent scale-up, only 15% of HIVexposed infants had access to early infant diagnosis and one-third of infants diagnosed of HIV did not receive appropriate antiretroviral treatment in 2011. Our results re-emphasize the crucial role of early infant diagnosis in order to initiate therapy for infected infants given their rapid progression [bib_ref] Early antiretroviral therapy and mortality among HIV-infected infants, Violari [/bib_ref]. LBW was previously associated with infant death between 8 weeks and 6 months of life [bib_ref] Timing of mother-to-child transmission of HIV-1 and infant mortality in the first..., Zijenah [/bib_ref] , post-neonatal death (29-365 days) and infant mortality [bib_ref] Association between low birth weight and infant mortality in children born to..., Wei [/bib_ref] among HIV-infected children as well as among HIV-exposed uninfected [bib_ref] The contribution of low birth weight to infant mortality and childhood morbidity, Mccormick [/bib_ref]. Among perinatally-infected infants, the effect of LBW on infant survival dominates the neonatal period but persists thereafter even once HIVrelated disease contributes to infant mortality. In our study, high maternal viral load and low hemoglobin were the most significant predictors of LBW and preterm birth as observed in other studies [bib_ref] Impact of maternal HIV infection on obstetrical and early neonatal outcome, Braddick [/bib_ref] [bib_ref] Determinants of low birth weight among HIV-infected pregnant women in Tanzania, Dreyfuss [/bib_ref] [bib_ref] Nutritional indicators of adverse pregnancy outcomes and mother-to-child transmission of HIV among..., Mehta [/bib_ref]. Advanced maternal disease seems to indirectly affect early infant survival via the risk of LBW and premature birth. Thus to complement antiretroviral interventions, other interventions such as providing cotrimoxazole [bib_ref] Reduction in preterm delivery and neonatal mortality after the introduction of antenatal..., Walter [/bib_ref] or multivitamin supplements during pregnancy [bib_ref] Randomised trial of effects of vitamin supplements on pregnancy outcomes and T..., Fawzi [/bib_ref] may need to be considered to improve overall birth outcomes.
Early breastfeeding cessation was a significant risk factor for both neonatal and early mortality. Breastfeeding has a significant role in infant survival for all infants including those born to HIV-infected women [bib_ref] Reduced mortality associated with breastfeeding-acquired HIV infection and breast-feeding among HIV-infected children..., Fox [/bib_ref] [bib_ref] Effects of early, abrupt weaning on HIV-free survival of children in Zambia, Kuhn [/bib_ref] [bib_ref] Elevations in mortality associated with weaning persist into the second year of..., Kuhn [/bib_ref]. Maternal death was also related to infant mortality and is closely related to cessation of breastfeeding. However, we found breastfeeding to be a strong protector of infant survival independent of confounding effects of maternal health and survival. Some social factors significantly affected infant survival. Infants with more than one sibling aged 5-16 years had decreased rates of early mortality. This may reflect families with greater birth spacing or substantial care provided by an older sibling. Teenage pregnancy was associated with an increased risk of neonatal and early mortality. In the general population, some studies have reported adverse associations between teenage pregnancy and perinatal mortality, largely attributable to preterm birth [bib_ref] Teenage pregnancies and risk of late fetal death and infant mortality, Olausson [/bib_ref] [bib_ref] Birth outcomes in teenage pregnancies, Gilbert [/bib_ref] [bib_ref] Increased risks of neonatal and postneonatal mortality associated with teenage pregnancy had..., Chen [/bib_ref] , while others have not [bib_ref] Teenage pregnancy and risk of adverse perinatal outcomes associated with first and..., Smith [/bib_ref]. In our study, maternal age <20 years was associated with neonatal mortality independent of preterm birth.
The study has several limitations. First, most of study participants were enrolled into the study during second or third trimester thus miscarriages and stillbirths occurring in earlier gestational ages are excluded resulting in underestimated rates of these outcomes. Our study thus has limited capacity to comment on the predictors of early pregnancy loss. However, most early pregnancy loss is due to chromosomal abnormalities and is unlikely to be influenced by maternal HIV status. Second, distinguishing between miscarriage and stillbirth is difficult particularly with inaccuracies in estimates of gestational age. Nevertheless, similar factors were associated with both of these adverse outcomes hence the consequence of misclassification seems to be limited. Third, HIV status could not be determined for some infants. However, we examined the extent of bias in sensitivity analyses which suggested that these exclusions did not appreciably affect the results. Finally, the cut-off of 70 days might have been too early to see any adverse effects of IP/ePP infection but this analysis aimed to examine how rapidly intrauterine and perinatally-acquired HIV infection affect infant survival.
# Conclusion
Women with more advanced HIV disease had a higher risk of pregnancy loss and perinatal mortality. Advanced maternal HIV disease affected infant health and survival directly via the risk of HIV transmission and indirectly via LBW and prematurity. The effect of intrauterine infection in the infant was apparent by 70 days but excess neonatal mortality was primarily attributed to LBW and preterm birth independent of infant HIV status. Interventions to improve maternal health as well as pregnancy outcomes for HIV-infected women may be necessary to complement the currently recommended prophylactic and therapeutic antiretroviral regimens.
[table] Table 1: Frequencies of adverse pregnancy outcomes among 1229 HIV-infected pregnant women recruited in Lusaka, Zambia [/table]
[table] Table 2: Risk factors for miscarriage and stillbirth among a cohort of 1229 HIV-infected women a [/table]
[table] Table 3: Risk factors for low birth weight (<2500g) and preterm birth among 1132 live-born infants a [/table]
[table] Table 4: Risk factors for neonatal mortality (<28 days) and early mortality (<70 days) among 1132 live-born infants a To consider whether risk factors for neonatal and early mortality were modified by infant HIV status, we stratified the analysis by infant HIV status. In multivariable model restricting to 128 infected infants, only LBW was associated with neonatal mortality (HR = 16.63; 95% 'CI 1.73-160.16) and LBW (AHR = 16.13; 95% CI '4.23- [/table]
[table] Table 5: Multivariate analysis of risk factors for neonatal and early mortality a [/table]
[table] Table 6: Sensitivity analyses for the 50 infants with unknown HIV status a Assume all of the 50 infants as NI at least for 42 days. b Assume all of the 50 infants infected by IU transmission. c Assume all of the 50 infants infected by IP/ePP transmission. d HIV status of the 50 infants was imputed based on the predicted probability of HIV transmission. e Adjusted for stopping breastfeeding, LBW, and preterm birth. f Adjusted for stopping breastfeeding, LBW, and 2+ children (5-16 aged). [/table]
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Seminal vesicle mass: An unusual site for metastatic hepatocellular carcinoma after orthotopic liver transplant
A B S T R A C TMetastasis of hepatocellular carcinoma (HCC) to the seminal vesicle is extraordinarily rare, with only two other cases reported in the literature. Herein we present the first documented case of a seminal vesicle as the initial site of solitary metastasis in a patient with a history of liver transplantation for HCC. We aim to provide more information regarding the disease process, histopathology, and management strategy.
# Introduction
Primary seminal vesicle tumors are extremely rare, with only a few cases reported in literature. Seminal vesicle malignancies more commonly result from contiguous invasion by tumors originating from adjacent organs such as prostate, bladder, and rectum. Thus far in the literature, there are only two reported cases that describe metastasis of hepatocellular carcinoma (HCC) to the seminal vesicle. [bib_ref] Seminal vesicle metastasis after partial hepatectomy for hepatocellular carcinoma, Gong [/bib_ref] [bib_ref] Seminal vesicle metastasis after liver transplantation for hepatocellular carcinoma: a case report, Shen [/bib_ref] Given that the most common sites of extrahepatic metastases occur in the lung, adrenals, bone, or lymph nodes, the fact that the seminal vesicle is the site of initial metastasis for a primary liver cancer is unheard of. [bib_ref] Hepatocellular carcinoma metastatic to the orbit: a case report, Oida [/bib_ref] Herein we present the first documented case of a seminal vesicle as the initial site of metastasis in a patient with a history of liver transplantation for HCC. Given that the seminal vesicles are an unusual site of metastasis for HCC, we aim to provide more information regarding disease process, histopathology, and management strategy.
## Case presentation
A 55-year old Caucasian male with a 5-year history of orthotopic liver transplant because of HCC presented to our department in October 2019 with new-onset weight loss, fatigue, and rectal pain of one-month duration. CT revealed a new 2.1 cm hyperdense nodule adjacent to the left seminal vesicle [fig_ref] Figure 1: Initial pelvic CT scan demonstrates 2 [/fig_ref]. His medical and surgical history was remarkable for hepatitis C status post treatment and alcoholic cirrhosis complicated by the development of HCC, status post radiofrequency ablation, chemoembolization x4, and liver transplantation. Social history included a 40 pack-year smoking history and significant alcohol use. Physical exam was within normal limits and the seminal vesicle mass was not palpable on rectal exam. Pelvic MRI revealed an indeterminate 2.1 × 2x2 cm enhancing nodule posterior to the left seminal vesicle [fig_ref] Figure 2: A, BSerial pre-operative axial pelvic MRI scans [/fig_ref]. CT-guided core needle biopsy was non-diagnostic. His lesion was followed with a repeat pelvic MRI 4 months later, which showed an interval increase of the mass to 2.5 × 2.2 × 2.8 cm [fig_ref] Figure 2: A, BSerial pre-operative axial pelvic MRI scans [/fig_ref]. Robotic exploration was performed for diagnostic purposes. Gelatinous material came from the mass as soon as the peritoneum was incised. The mass had poorly defined margins and appeared to be infiltrating the posterior bladder wall. The dissection was very difficult as in most cases of tumors with the presumptive mechanism of dissemination of peritoneal "drop metastases". After ureterolysis was performed, the left vas deferens was surrounded by thick tissue. The seminal vesicle and vas deferens were dissected en bloc. Histopathologic examination revealed metastatic carcinoma involving the soft tissue adjacent to the seminal vesicle and extending to margins. The cells were positive for Hepatocyte Paraffin-1 and Arginase-1 and negative for Glypican-3-consistent with metastasis from the patient's primary HCC [fig_ref] Figure 3: A, BHistology slides of seminal vesicle and hepatic masses [/fig_ref]. CT chest, abdomen, and pelvis 5 weeks post-operatively showed a 26 × 20 mm residual soft tissue density within the resection bed with mild tethering of the anterior wall of the rectum towards it (not shown). No other recurrent or metastatic lesions were identified. The patient was advised to undergo external beam radiation therapy to the seminal vesicles in addition to concurrent chemotherapy with capecitabine for 5.5 weeks.
# Discussion
HCC metastasis to the seminal vesicles after liver transplant is very rare, with only one other case reported in a patient with already documented metastases. [bib_ref] Seminal vesicle metastasis after liver transplantation for hepatocellular carcinoma: a case report, Shen [/bib_ref] In this case, the patient had a history of HCC status post orthotopic liver transplant. His preoperative imaging did not reveal any abnormalities in the prostate, bladder, or rectum; his rectal exam was within normal limits, and a recent colonoscopy was normal. This case underscores the importance of conducting a comprehensive history and physical examination when evaluating patients with seminal vesicle masses given the nature and etiology of seminal vesicle tumors.
Imaging also plays an important role in the evaluation of a seminal vesicle mass, with MRI being the most accurate tool in view of its excellent tissue contrast and multiplanar technology. [bib_ref] Imaging of the seminal vesicle and vas deferens, Kim [/bib_ref] Another benefit of MRI is its accuracy in identifying seminal vesicle invasion by adjacent organs. Despite recent advances in imaging techniques, there are no reliable features that can differentiate primary from secondary malignancy on MRI, making tissue sampling often necessary for an accurate diagnosis. [bib_ref] Lesions of the seminal vesicles and their MRI characteristics, Reddy [/bib_ref] Because our patient had an inconclusive MRI and nondiagnostic biopsy, he underwent robotic-assisted seminal vesiculectomy for diagnostic and therapeutic purposes. An intraoperative frozen section suggested that the patient's malignancy was metastatic from his HCC. After consultation with medical oncology, a decision was made to resect the seminal vesicle and vas deferens en bloc instead of pursuing a more aggressive approach (ie. partial cystectomy with ureteral reimplantation or radical cystoprostatectomy). The preoperative evaluation of a seminal vesicle mass should focus on distinguishing between benign and malignant lesions since the nature of the lesion will guide surgical planning. Likewise, intraoperative frozen pathology sections also have an important role in managing seminal vesicle masses as they can help distinguish primary versus secondary etiologies and guide treatment strategies. The presumptive mechanism of metastasis of HCC to the seminal vesicle is peritoneal implant by "drop metastasis".
Given that metastasis of HCC to the seminal vesicles is very rare, there are no guidelines for the workup or management of this disease. Surgical intervention can have a diagnostic and therapeutic role in treatment, especially if the patient's metastasis is confined to one site and clear margins are obtained. Beyond surgical management, however, there is limited data regarding the role of postoperative antineoplastics. In our case, postoperative management with radiation therapy and concurrent chemotherapy with capecitabine was advised given his young age, excellent performance status, long disease-free interval prior to this event, and small volume metastatic disease. Patients with metastatic HCC ultimately have a poor prognosis, so shared decision making should be employed to come up with a plan in accordance with the patient's wishes.
# Conclusion
Seminal vesicle metastasis from HCC is rare. Thorough historytaking, physical examination, abdominopelvic MRI, and immunohistochemical staining are helpful in diagnosis. Surgery has a diagnostic and potentially therapeutic role in management; however, these patients ultimately have poor prognoses. More data is required to determine the role of postoperative radiation therapy with concurrent antineoplastics.
## Consent
Informed consent was obtained from patient verbally over telephone by author JTX.
# Funding
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
## Declaration of competing interest
None.
[fig] Figure 1: Initial pelvic CT scan demonstrates 2.1 cm hyperdense nodule adjacent to the left seminal vesicle. [/fig]
[fig] Figure 2: A, BSerial pre-operative axial pelvic MRI scans. (A) Initial pelvic MRI reveals indeterminate 2.1 × 2 × 2 cm enhancing nodule posterior to the left seminal vesicle with no definite restricted diffusion. (B) Subsequent MRI after four months shows a slight interval increase in the size of the 2.5 × 2.2 × 2.8 cm oval mass. [/fig]
[fig] Figure 3: A, BHistology slides of seminal vesicle and hepatic masses. (A) Microscopic view of seminal vesicle mass, 400x. (B) Microscopic view of hepatic mass, 400x. [/fig]
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Association of Electronic Health Record Design and Use Factors With Clinician Stress and Burnout
IMPORTANCE Many believe a major cause of the epidemic of clinician burnout is poorly designed electronic health records (EHRs).OBJECTIVES To determine which EHR design and use factors are associated with clinician stress and burnout and to identify other sources that contribute to this problem.DESIGN, SETTING, AND PARTICIPANTSThis survey study of 282 ambulatory primary care and subspecialty clinicians from 3 institutions measured stress and burnout, opinions on EHR design and use factors, and helpful coping strategies. Linear and logistic regressions were used to estimate associations of work conditions with stress on a continuous scale and burnout as a binary outcome from an ordered categorical scale. The survey was MAIN OUTCOMES AND MEASURES Clinician stress and burnout as measured with validated questions, the EHR design and use factors identified by clinicians as most associated with stress and burnout, and measures of clinician working conditions. RESULTS Of 640 clinicians, 282 (44.1%) responded. Of these, 241 (85.5%) were physicians, 160 (56.7%) were women, and 193 (68.4%) worked in primary care. The most prevalent concerns about EHR design and use were excessive data entry requirements (245 [86.9%]), long cut-and-pasted notes (212 [75.2%]), inaccessibility of information from multiple institutions (206 [73.1%]), notes geared toward billing (206 [73.1%]), interference with work-life balance (178 [63.1%]), and problems with posture (144 [51.1%]) and pain (134 [47.5%]) attributed to the use of EHRs. Overall, EHR design and use factors accounted for 12.5% of variance in measures of stress and 6.8% of variance in measures of burnout. Work conditions, including EHR use and design factors, accounted for 58.1% of variance in stress; key work conditions were office atmospheres (β = 1.26; P < .001), control of workload (for optimal control: β = −7.86; P < .001), and physical symptoms attributed to EHR use (β = 1.29; P < .001). Work conditions accounted for 36.2% of variance in burnout, where challenges included chaos (adjusted odds ratio, 1.39; 95% CI, 1.10-1.75; P = .006) and physical symptoms perceived to be from EHR use (adjusted odds ratio, 2.01; 95% CI, 1.48-2.74; P < .001). Coping strategies were associated with only 2.4% of the variability in stress and 1.7% of the variability in burnout. CONCLUSIONS AND RELEVANCE Although EHR design and use factors are associated with clinician stress and burnout, other challenges, such as chaotic clinic atmospheres and workload control, explain considerably more of the variance in these adverse clinician outcomes. Question Which electronic health record (EHR) design and use factors are associated with clinician stress and burnout? Findings In this survey study of 282 clinicians, clinician stress and burnout were associated with 7 EHR design and use factors. These 7 plus 2 other design and use factors collectively accounted for a modest amount of the variance in stress (12.5%) and burnout (6.8%); models incorporating other work conditions (such as chaotic work atmosphere and workload control) accounted for considerably more of the variance in stress (58.1%) and burnout (36.2%).Meaning While EHR design and use factors may appropriately be targeted by health systems and EHR designers to address stress and burnout, other non-EHR issues, especially clinician work conditions, appear to play a substantial role in adverse clinician outcomes.
# Introduction
The adoption of the electronic health record (EHR) has occurred alongside the dramatic and troubling rise in clinician stress and burnout.This association has fueled the debate over the extent to which EHRs are associated with the epidemic of clinician stress and burnout. Technostress (ie, the stress related to technological tools in numerous industries) is real, 4 but the degree to which it is a factor in medicine is largely unknown.
The introduction of EHRs has resulted in shifting many clerical tasks to clinicians (eg, billing, coding, and quality control) as well as creating new tasks to be performed during clinical encounters (eg, data entry, computerized decision support, computerized order entry, and electronic prescribing). These new tasks have increased the cognitive and physical load on the clinician in many ways.For example, e-prescribing, which has benefits, has also created an additional burden by requiring clinicians to know where to route prescriptions at the time they prescribe. This may be a relatively small burden, but repeated multiple times per day and added to the myriad other tasks shifted to clinicians, these technology-enabled tasks have considerably increased clinician workload.
In fact, an entirely new medical scribe industry has arisen in order to ameliorate the additional workload.We designed this study (Minimizing Stress, Maximizing Success of the Electronic Health Record) to identify the relative contribution of aggregated EHR burdens compared with other burdens (ie, workplace chaos, control of workload) associated with clinician stress and burnout. This work is based on a conceptual framework derived from prior work .Our hypothesis was that EHR-associated stress adds to overall stress and could lead to burnout-which may play a role in the quality of patient care. In this study, we aim to understand which EHR design and use factors are associated with stress and burnout. The potentially challenging EHR design and use factors included in the survey instrument were identified through physician focus groups conducted in the first phase of the study.The design and use factors studied were intentionally limited to those over which clinicians and their institutions might have some control. This in no way minimizes other societal factors, such as governmental regulation and malpractice, that could be associated with clinician stress and burnout.This survey phase of our study quantifies the association of these EHR design and use factors with clinician stress and burnout to address the following questions:what specific EHR design and use factors are most strongly associated with clinician stress and burnout? (2) What amount of overall stress and burnout is associated with EHRs? And,what coping strategies or organizational solutions did respondents feel are important in addressing stress and burnout?
## Figure. conceptual framework of association of work conditions and electronic health record (ehr) design and use factors with clinician outcomes
# Methods
## Identification of challenging ehr design and use factors
The methods for this study have been previously reported.In brief, physician focus groups at 3 institutions (Stanford Hospital and Clinics, Stanford, California; University of New Mexico, Albuquerque; and Centura Health Physician Group, Westminster, Colorado) identified EHR design and use factors that were perceived as successful and those that were associated with user stress, burnout, or unintended physical symptoms. We also identified commonly used coping strategies by the clinicians.
## Survey and sampling
The EHR design and use factors identified in prior clinician focus groups informed the design of the survey instrument, which is freely available.The instrument included questions from previously validated instruments to measure stress, burnout, and other challenges identified by the Physician Worklife Survey,The survey's design attempted to determine the following: (1) perceived EHR successes,EHR design and use factors associated with clinician stress and burnout, (3) perceived adverse personal outcomes (eg pain or anxiety), (4) things that could improve the EHR experience (eg, greater staff support, scribes, or fewer clicks per task), and (5) coping strategies (eg, exercise or setting boundaries). We sampled clinicians (physicians and advanced practice clinicians, including nurse practitioners and physician assistants) at 3 institutions from 5 disciplines: general internal medicine, medical subspecialties, general pediatrics, pediatric subspecialties, and family medicine.
We excluded residents, as we thought they could have dissimilar experiences of stress and burnout than practicing clinicians. We determined respondent stress levels using the 4-item validated measures from Motowidlo, 14 a continuous measure that ranges from 4 to 20, and burnout using the single-item validated measure from the Physician Worklife Study, in which a score of 3 or more indicates burnout.While a binary approach to burnout has been controversial, 22,23 this measure has been used and validated in many settings and among thousands of respondents for 20 years, and it is associated with adverse work conditions and adverse clinician outcomes, such as intent to leave the practice. We ran additional analyses using the 5-choice measure of burnout as an ordered categorical (as opposed to binary) outcome and found no substantive differences between the 2 methods.
# Statistical analysis
Answers to survey questions were analyzed as standard summary statistics. We reported continuous variables as mean and SD and categorical variables as number of respondents and percentages of total sample.
Linear regression was used to determine the association of focus group-identified variables (eg, work conditions, EHR design and use factors, and coping strategies) with clinician-reported stress, which we scored according to the Motowidlo 4-item measure,and burnout. β was used to estimate the magnitude and direction of association, and it was calculated using the least-square estimation technique. We used logistic regression with stepwise selection, which is a combination of the forward and backward selection techniques, to estimate the association of focus group-identified variables with the odds of clinician-reported burnout, which we measured as a binary outcome based on a single question (with burnout representing endorsement of any choice with the word burnout in it).We used construct variables created to summarize the associations of variables within the same domain with stress and burnout. (These showed that the models were well calibrated.) Finally, we performed a statistical factor analysis using the varimax rotation method on 9 EHR design and use items to summarize the association of EHRs with stress and burnout. We used SAS version 9.4 (SAS Institute, Inc) for all analyses. Statistical significance was set at P < .05, and all tests were 2-tailed. definitely likely to leave their practices within 2 years.
# Results
## Sample and work-life balance description
## Jama network open | health informatics
## Association of ehr use and design factors with stress and burnout
The EHR design and use factors significantly associated with high clinician stress were information overload (β = 0.37; P < .001), slow system response times (β = 0.42; P < .001), excessive data entry
## Other factors associated with stress and burnout
Factors not related to EHRs associated with high levels of variance in stress were office atmospheres, these variables, along with the EHR design and use factors listed in
## Jama network open | health informatics
## Factor analysis of ehr stress items
We performed a statistical factor analysis using the varimax rotation method on the 9 EHR design and use factors listed in. We found that the first 2 statistical factors from the factor analysis accounted for 52.2% of the variability in EHR design and use items. We characterize these 2 factors as follows: (1) interference with patient care (eg, note bloat, interference with patient-clinician relationships, and notes geared toward billing) and (2) inefficient systems (eg, slow system response times, inability to navigate the system quickly, and excessive data entry). Thus, more than half of the variance in EHR issues associated with clinician stress and burnout stemmed from interference with patient care and inefficient EHR systems.
# Discussion
In this cross-sectional survey of 282 clinicians from 3 health systems, we identified 7 EHR design and use factors associated with high stress and burnout. These were information overload, slow system response times, excessive data entry, inability to navigate the system quickly, note bloat, interference with the patient-clinician relationship, fear of missing something, and notes geared toward billing. While previous studies have identified several of these EHR design and use items as challenging to clinicians,we believe this study is the first to show an association between these factors and objectively validated stress and burnout scales.
In this study, 45.0% of participants described symptoms of burnout, consistent with the findings of the national survey by Shanafelt et al 2 in which 44% of physicians reported at least 1 symptom of burnout. The amounts of variation in stress and burnout associated with the EHR design and use factors listed inwere 12.5% and 6.8%, respectively. Thus, other sources of burnout aside from the EHR (such as lack of control of workload, chaotic environments, lack of attention to work-life balance, and ineffective teamwork) will also need to be addressed as medical practices seek to reduce burnout. Information overload may be associated with EHR design in which too much clinically unnecessary information is displayed. The aviation industry has a user interface design philosophy called quiet dark, where information is not displayed until something goes wrong or needs the pilot's attention. In other words, the default state of all indicator lights is off during normal conditions.
Applying this philosophy to EHR design could potentially reduce the amount of unnecessary data displayed based on particular users' need and context, reducing the information overload problem.
Arguably, the current state of EHR design is loud bright, where virtually all information, normal or otherwise, appears in relatively the same manner regardless of its importance to the clinician or patient. Although abnormal results from laboratory tests are highlighted, all normal values are typically displayed and occupy the same amount of space and are given the same prominence as abnormal results. Given the proliferation of standardized templates as a time-saving tool for data entry, the amount of unnecessary, repetitive, normal information (ie, note bloat) is increasing vs a design where an economy of information relevant to the patient's current needs and context is used. 29
The data entry problem has created the scribe movement and produced promising results, at least in terms of clinician and patient satisfaction.However, scribes only help with data entry during office visits and not with EHR tasks at other times and in other venues. A more comprehensive approach is to use specially trained medical assistants (MAs) to relieve the clinician from clinic tasks (eg, responding to routine in-basket messages, refilling some prescriptions per protocol, completing paperwork). Before the clinician meets the patient, the MA completes prework (eg, medication reconciliation, review of systems, documentation of chief concern, and any protocolized clinical measurements, such as peak flows or pulse oximetry). The MA scribes during the clinician encounter, and after the clinician leaves the room, the MA can review the plan of care, deliver patient education, process referral requests, and schedule follow-up appointments.Some of the troublesome EHR design and use factors, such as the inability to navigate the system quickly, are attributable to computer-human interaction problems. In fact, most of the current EHR user interface designs are still based on 2-dimensional paper metaphors (eg, tabs, flowsheets, tables, and forms) and do not take advantage of the potential of graphics capabilities now in the most basic computers.More research to determine what display metaphors beyond paper are most efficient could help. Complaints of interference with the patient-clinician relationship is evidence that clinicians are troubled by their excessive focus on the screen rather than the patient.
While most studies have shown the presence of the EHR in the exam room does not adversely affect patient satisfaction,clinicians feel that EHRs requiring clinically irrelevant data entry take away from their relationships with their patients.Our study shows that this is significantly associated with clinician stress and burnout.
The proportion of clinicians reporting pain (47.5%) and posture issues (51.1%) attributed to EHR use was high. Ergonomics are rarely addressed in most clinical settings. Clinicians often must work at several workstations, with different heights and seat structures. Collaboration with employee health groups skilled at ergonomics could potentially have a substantive effect on the health outcomes of our clinician workforce.This is an area ripe for further quality improvement studies.
Coping strategies clinicians suggested to reduce EHR-associated stress included exercise (used by 68.1% of our sample), verbally discussing issues with other clinicians (68.8%), and setting boundaries for work while at home (57.1%). Setting boundaries, exercise, and taking breaks were significantly associated with reductions in overall stress and burnout and may be useful components to incorporate into stress reduction interventions. It is not clear how many of these strategies clinicians actually used or how effective they were at using them.
# Strengths and limitations
The strengths of this study include surveying a diverse group of clinicians, including academic, community-based, and rural institutions and practices, physicians and advanced practice clinicians, and a mix of specialists and nonspecialty ambulatory care clinicians. In addition, the list of the EHR design and use factors the clinicians rated in the survey was defined by clinicians in multi-institutional focus groups.The survey response rate (44.1%) was reasonable for large clinician-based studies with no financial incentive. The design of the instrument included questions previously validated in studies of physicians about stress and burnout.
This study has limitations, including its cross-sectional nature and the use of self-reported metrics. One needs to consider response bias, given the 44.1% response rate. The relatively modest sample size limits validity. As respondents came from only 3 institutions, these results may not be more widely generalizable. The mapping of the paper instrument's Likert scales to the REDCap slider bars scale may have introduced some bias. Despite using validated instruments to measure burnout and stress, the survey relied on the respondents' own definitions. Self-reported metrics may underrepresent the numbers at risk. As Knox et al 37 found, a self-defined, single-item burnout measure identified significantly fewer physicians most at risk of burning out compared with the Maslach Burnout Inventory. All respondents were grouped together for this analysis, which does not account for possible intragroup differences, such as between physicians and advanced practice clinicians. 37
# Conclusions
Stress and burnout associated with EHRs is prevalent and may be at least partly remediable at the local level. The issues identified in our list of EHR-associated challenges may provide designers, government regulators, and clinical leaders with targets for improvement of EHR design. Other work conditions are associated with stress and burnout in clinicians and deserve equal attention. |
Newly-diagnosed diabetes and admission hyperglycemia predict COVID-19 severity by aggravating respiratory deterioration
[bib_ref] Diabetes mellitus, fasting glucose, and risk of cause-specific death, Rao Kondapally Seshasai [/bib_ref]
# Introduction
Diabetes worsens the outcome of virtually any acute or chronic medical condition, resulting in a shortened life expectancy [bib_ref] Diabetes mellitus, fasting glucose, and risk of cause-specific death, Rao Kondapally Seshasai [/bib_ref]. Mortality from infectious disease is also increased in patients with diabetes, especially for sepsis and pneumonia [bib_ref] Mortality from infectious diseases in diabetes, Zoppini [/bib_ref]. Since the very beginning of the SARS-CoV-2 pandemic, diabetes emerged as one of the most common comorbidities and a potential driver of poor outcomes [bib_ref] Practical recommendations for the management of diabetes in patients with COVID-19, Bornstein [/bib_ref]. The prevalence of diabetes was higher among patients hospitalized for COVID-19 who were admitted to the intensive care unit (ICU) or died [bib_ref] Clinical characteristics of coronavirus disease 2019 in China, Guan [/bib_ref]. Meta-analyses of studies reporting the characteristics of patients according to COVID-19 severity in China found that diabetes conferred a 2-3 fold increased rate of poor disease outcome [bib_ref] Diabetic patients with COVID-19 infection are at higher risk of ICU admission..., Roncon [/bib_ref] [bib_ref] Prevalence and impact of diabetes among people infected with SARS-CoV-2, Fadini [/bib_ref]. These findings are in line with the available literature on the adverse prognostic impact of diabetes on other viral infections, including influenza [bib_ref] Characteristics and outcome of viral pneumonia caused by influenza and Middle East..., Al-Baadani [/bib_ref] [bib_ref] Clinical courses and outcomes of hospitalized adult patients with seasonal influenza in..., Hong [/bib_ref].
It remains unclear whether diabetes drove such excess risk independently from confounders, including its complications and comorbidities. It is conceivable that patients with diabetes experienced a worse COVID-19 outcome because of multiple organ damage due to micro-and macrovascular disease [bib_ref] The contribution of diabetic micro-angiopathy to adverse outcomes in COVID-19, Whyte [/bib_ref]. On the other side, whether the impact of diabetes on COVID-19 outcome depends on glucose control is less appreciated. In a study conducted on 7337 Chinese patients, diabetes was associated with higher mortality, but patients with good glycemic control had lower mortality compared to those with poorer control [bib_ref] Association of blood glucose control and outcomes in patients with COVID-19 and..., Zhu [/bib_ref]. Optimal glycemic management can therefore be crucial to improve COVID-19 outcome [bib_ref] Outcomes in patients with hyperglycemia affected by Covid-19: can we do more..., Sardu [/bib_ref]. Finally, new-onset diabetes has been reported during COVID-19 [bib_ref] COVID-19 symptoms masking inaugural ketoacidosis of type 1 diabetes, Potier [/bib_ref] [bib_ref] Diabetic ketoacidosis precipitated by Covid-19 in a patient with newly diagnosed diabetes..., Chee [/bib_ref] [bib_ref] COVID-19 infection may cause ketosis and ketoacidosis, Li [/bib_ref] , but its impact on disease outcome has not been assessed. In patients with acute medical conditions, such as acute myocardial infarction, newly-detected diabetes has an adverse prognostic effect [bib_ref] Diabetes known or newly detected, but not impaired glucose regulation, has a..., Lenzen [/bib_ref] [bib_ref] Cardiovascular outcomes using doxazosin vs. chlorthalidone for the treatment of hypertension in..., Barzilay [/bib_ref]. Of note, newlydiagnosed diabetes is often linked with occult organ damage that shortens survival [bib_ref] Prognostic significance of silent myocardial infarction in newly diagnosed type 2 diabetes..., Davis [/bib_ref]. In addition, stress hyperglycemia has been known for decades to drive an exaggerated inflammatory response in critically-ill individuals [bib_ref] Stress-hyperglycemia, insulin and immunomodulation in sepsis, Marik [/bib_ref].
In the present study, we investigated the role of preexisting diabetes, newly-diagnosed diabetes, and admission glucose levels on the outcome of patients hospitalized for COVID-19. We eventually explored which were the mediators of a poor outcome associated with diabetes.
## 2.
Subjects, materials and methods
## Study design
This retrospective study was conducted by collecting anonymized patient's data from electronic medical records. The protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki. In agreement with national regulation on retrospective studies, the protocol was notified to the local ethical committee (no. 0031264) and the need for patient's informed consent was waived.
## Data collection
We retrieved data on all consecutive patients hospitalized for COVID-19 between February 21st and April 20th 2020 at the University Hospital of Padova, which is located at the center of one of the first SARS-CoV-2 outbreaks in Northern Italy.
We screened records of all patients admitted to the Hospital with a positive PCR test for SARS-CoV-2 on upper or lower airway sample. Pre-existing diabetes was defined based on selfreported history, prior electronic medical records reporting a diagnosis of diabetes, or ongoing therapy with glucoselowering medications. Newly-diagnosed diabetes was defined by a HbA1c value of 48 mmol/mol (6.5%) or higher; in the absence of an HbA1c determination, a random glucose level of 11.1 mmol/l (200 mg/dl) or higher, accompanied by signs and symptoms of hyperglycemia was considered diagnostic. For all patients, we recorded the following information: demographics (age, sex), concomitant cardiovascular risk factors (smoke, hypertension, dyslipidemia), comorbidities (chronic obstructive pulmonary disease and history of cancer), complications (cardiovascular disease and microangiopathy), presence or absence of COVID-19 related pneumonia or interstitial lung disease (ILD), ongoing therapies before hospitalization. We collected information on symptoms of COVID-19 upon admission (time from onset of symptoms to hospitalization; presence of fever, cough, dyspnea, and gastrointestinal symptoms). Vital signs were recorded at admission and at their worst timepoint: systolic and diastolic blood pressure, heart and respiratory rate, oxygen saturation, PaO 2 /FiO 2 ratio. Key laboratory exams included: fasting plasma glucose, HbA1c, lipid profile (total cholesterol, HDL cholesterol, triglycerides, and calculated LDL cholesterol), and serum creatinine for calculation of estimated glomerular filtration rate (eGFR) using the CKD-EPI equation. We also collected admission levels and worse levels of the following biomarkers related to inflammation (white blood cell count [WBC], C-reactive protein [CRP], IL-6, pro-calcitonin), hematology and coagulation (hemoglobin, lymphocytes, platelets, D-dimer) and tissue injury (liver enzymes, troponin I, lactate). We reported the worst vital signs or biomarkers as the worst value measured during the entire hospital stay, regardless of whether it occurred at admission or during any other time of hospitalization. In some cases, worst values could coincide with admission values if admission values were the worst among all values recorded during the hospital stay. The following inhospital treatments were recorded: low-flow and high-flow oxygen, non-invasive ventilation, intubation, use of specific drugs, such as lopinavir/ritonavir, azithromycin, other antibiotics, remdesivir, chloroquine/hydroxychloroquine, glucocorticoids, tocilizumab.
## Outcome definition
The primary outcome was a composite of admission to the ICU (including all subjects needing mechanical ventilation) or death. Death from any cause, was considered as a separate outcome. Among those who were discharged alive, time to discharge was considered as additional secondary outcomes. Because duration of hospitalization was highly skewed and sometimes driven by non-clinical reasons (e.g. patients being unable to return to home because lacking social support), observation was censored at 30 days.
# Statistical analysis
Continuous variables are reported as mean ± standard deviation (SD) or as median (inter-quartile range), while categorical variables have been reported has percentages. Betweengroup differences in clinical characteristics were evaluated with Student t-test or chi-Square for continuous and categorical variables, respectively. The association between diabetes, glucose levels and other variables of interest with categorical outcomes was evaluated with robust-errorvariance Poisson regression models [bib_ref] A modified poisson regression approach to prospective studies with binary data, Zou [/bib_ref]. In order to identify possible confounding factors, these associations were tested in unadjusted models and in two different multivariable adjusted (MVA) models with increasing complexity. Model 1 (MVA1): adjusted for sex and age. Model 2 (MVA2): adjusted for sex, age and pre-existing conditions or pre-hospital medications associated with COVID-19 severity in MVA1. In order to test the MVA, full dataset of variables were needed, thus missing data were handled by means of multiple imputation (MI). MI was performed with a fully conditional specification (FCS) algorithm [bib_ref] Multiple imputation by fully conditional specification for dealing with missing data in..., Liu [/bib_ref] obtaining 10 imputed datasets including only covariates with less than 60% of missing values. Outcome variables and main variables of interest (e.g. plasma glucose) were not imputed. Outcome analyses were performed on each imputed dataset and the pooled estimated effects are presented [bib_ref] Multiple imputation after 18+ Years, Rubin [/bib_ref]. Cox proportional hazard model was used to compare the patient's probability of being discharged alive. The differences between the association of pre-existing diabetes and newly-diagnosed diabetes with the primary and secondary outcomes were evaluated by means of Z score (Z score = (estimates 1 À esitmates 2 )/ p (SE 1 2 + SE 2 2 )). The differences in the association between glucose levels and primary outcome among subjects with or without diabetes was evaluated with the inclusion of an interaction term in the models. The association between fasting plasma glucose and the worst levels of clinical and laboratory parameters detected during hospital stay were represented graphically with scatter plots and described by Pearson' r correlation coefficient.
We evaluated which of these parameters explained the association between glucose levels and COVID-19 severity outcome (i.e. being mediators). To be consider a candidate mediator, variables needed to be independently associated with both the primary outcome (tested using Poisson regression with robust error variance) and with glucose levels (tested with linear regression). For those variables meeting these two conditions, we then quantified the possible mediation effect (in percentage) as the differences in the association between glucose and the primary outcome in the model adjusted by the candidate mediator (RR ADJUST ) as compared to the association in the model not adjusted by the candidate mediator (RR NOT-ADJUST ). Therefore the mediation analyses was evaluated as: mediation = 100 Â [ln(RR NOT-ADJUST ) À ln (RR ADJUST )]/ln(RR NOT-ADJUST ). Statistical analyses were conducted with a significance threshold of p < 0.05 and were done in SAS version 9.4 (TS1M4), GraphPad PRISM v8.3.0.
# Results
## Characteristics of the covid-19 population by diabetes status
We included 413 patients who tested positive for SARS-CoV-2 upon PCR analysis. Of these, 107 (25.6%) had diabetes (86 preexisting and 21 newly diagnosed). As compared to individuals without diabetes, those with diabetes were older, had a higher prevalence of hypertension, dyslipidemia, cardiovascular disease, and chronic kidney disease. In addition, pre-admission use of statins, antiplatelet drugs and beta-blockers were more common among subjects with diabetes . Symptoms at admission were similar as were laboratory findings (except for FPG and HbA1c). Participants with diabetes had more compromised respiratory function with lower PaO 2 /FiO 2 and higher respiratory rate [fig_ref] Table 2 -: Hospital treatments and outcomes according to diabetes status [/fig_ref]. In-hospital pharmacological treatments were similar in subjects with or without diabetes, except that diabetic patients more often received antibiotics other than macrolids [fig_ref] Table 2 -: Hospital treatments and outcomes according to diabetes status [/fig_ref]. shows patients' clinical characteristics further stratified by pre-existing versus newly-diagnosed diabetes status.
## Diabetes and severity of covid-19
Over a median observation time of 17 days (IQR 6-26), 102 patients (24.7%) showed a severe course, defined as the primary outcome, 48 of whom died (11.6%). In unadjusted analysis, presence of diabetes (including pre-existing and newlydiagnosed) compared to its absence was associated with a higher incidence of the primary outcome (37.4% vs 20.3%; RR 1.85; 95% C.I. 1.33-2.57; p < 0.001; . Several other pre-existing conditions were associated with COVID-19 severity, including hypertension, cardiovascular disease, atrial fibrillation, CKD and COPD [fig_ref] Table 2 -: Hospital treatments and outcomes according to diabetes status [/fig_ref]. Among prehospitalization treatments, use of ARBs, novel oral anticoagulants, and systemic glucocorticoids was more frequent among patients with the primary severity outcome [fig_ref] Table 2 -: Hospital treatments and outcomes according to diabetes status [/fig_ref]. Compared to absence of diabetes, newly diagnosed diabetes (RR 3.06; 95% C.I. 2.04-4.57) showed a stronger association with the primary outcome than pre-existing diabetes (RR 1.55, 95% C.I. 1.06-2.27). The difference in the RR for the primary outcome between pre-existing and newlydiagnosed diabetes was statistically significant (p = 0.004). In addition, higher FPG at admission was associated with COVID-19 severity, with an increase in relative risk of 21% (RR 1.21; 95% C.I. 1.11-1.32; p < 0.001) for each 2 mmol/L (36 mg/dl) increase in FPG. This association was stronger among individuals without diabetes than in those with diabetes (p for interaction = 0.003; After adjusting for age and sex, the magnitude of the association between diabetes (pre-existing and newly-diagnosed altogether) and COVID-19 severity remained significant (RR 1.49; 95% C.I. 1.07-2.09; p = 0.019; . Other variables d i a b e t e s r e s e a r c h a n d c l i n i c a l p r a c t i c e 1 6 8 ( 2 0 2 0 ) 1 0 8 3 7 4 -Characteristics of COVID-19 patients according to diabetes status. Data presented as mean (SD) or as percentage. In the column reporting data of all patients, data availability is also shown. Diabetes includes both pre-existing diabetes and newly-detected diabetes. [fig_ref] Table 2 -: Hospital treatments and outcomes according to diabetes status [/fig_ref]. The analysis was repeated splitting diabetes into preexisting and newly-diagnosed. The association of preexisting diabetes was attenuated and no longer significant, whereas the association between newly-diagnosed diabetes with the primary outcome remained statistically significant. The association between FPG and the primary outcome remained significant after age-and sex-adjustment and yet with a larger effect on outcome among non-diabetic than diabetic subjects .
## Independent predictors of critical covid-19 severity
We tested whether the associations of diabetes and FPG with COVID-19 severity was independent from additional confounders. As shown in , the association between diabetes and the primary outcome was confirmed in all models, including those with pre-existing comorbidities and med- ications used prior to hospitalization (RR 1.61; 95% C.I. 1.12-2.30; p = 0.010). Similarly, the association between FPG and the primary outcome was confirmed in all models , including those with pre-existing comorbidities and medications used prior to hospitalization (RR per each 2 mmol/l = 1.14; 95% C.I. 1.06-1.23; p < 0.001).
## Diabetes and covid-19 mortality
We found a similar mortality rate among those with or without diabetes [fig_ref] Table 2 -: Hospital treatments and outcomes according to diabetes status [/fig_ref]. In a time-toevent analysis, diabetes was associated with a 43% reduced probability of recovery after adjustment for age and sex (HR 0.58; 95% C.I. 0.43-0.77). Among survivors, the duration of hospitalization was significantly longer for patients with as compared to those without diabetes in unadjusted (+3.7 days; 95% C.I. 1.9-5.5, p < 0.001; [fig_ref] Table 2 -: Hospital treatments and outcomes according to diabetes status [/fig_ref] and age-and sex-adjusted models (+2.6 days; 95% C.I. 0.9-4.6, p = 0.003). For each 2 mmol/l higher FPG, there was a significant 19% lower probability of recovery (HR 0.82 95% C.I. 0.74-0.91; p < 0.001) and longer duration of hospitalization (+1.0 days; 95% C.I. 0.5-1.5; p < 0.001).
## Mediators of association between glucose and covid-19 severity
We found statistically significant correlations between admission glucose levels and most clinical-laboratory characteristics, recorded at their worst level, indicative of COVID-19 severity or progression, including hemodynamic, respiratory, hematologic, inflammatory, and tissue damage biomarkers [fig_ref] Figure 2 -: Correlation between admittance glucose levels and severity clinical variables [/fig_ref].
We then evaluated whether these correlations could partially explain (i.e. mediate) the association between FPG and -Forest plots of the association of diabetes/hyperglycemia on COVID-19 outcomes. Unadjusted (A) and age-and sexadjusted (B) analysis of the association between diabetes, as compared to no diabetes, split known diabetes versus newlydiagnosed diabetes and hyperglycemia (for each 2 mmol/l increase) with severe COVID-19. The primary severity outcome was admittance to the intensive care unit, or need of mechanical ventilation, or death (results of model 2 are detailed in , and not are graphically represented here since results were very similar to model 1).
COVID-19 severity outcome. We first identified clinicallaboratory characteristics (recorded at their worst level) significantly associated with COVID-19 severity (independently from age and sex). Then, we estimated what percentage of the effect of FPG on COVID-19 severity was explained by the association between FPG and these variables. As described in and represented graphically in [fig_ref] Figure 3 -: Mediators of the effects of hyperglycemia on COVID-19 outcomes [/fig_ref] , we found that a decline in respiratory function variables was the major determinant of the detrimental effect of hyperglycemia on COVID-19 severity.
# Discussion
In this study, we found that newly-detected diabetes and admission hyperglycemia were more strongly associated with COVID-19 severity outcome than pre-existing diabetes. A known diabetes status was more common among patients with severe COVID-19, defined by ICU admittance or death.
Known diabetes remained associated with poor COVID-19 outcome independently from age and sex, but not when adjusted for other baseline clinical variables, suggesting that such effect was mostly driven by concomitant factors and complications. On the other side, newly-detected diabetes remained associated with COVID-19 severity in fullyadjusted analyses. Upon a formal comparison, the association with the primary outcome was stronger for newlydiagnosed than for pre-existing diabetes. Admission glucose levels were closely related to most clinical and biochemical parameters of COVID-19 severity collected during hospitalization, including the PaO 2 /FiO 2 ratio. Remarkably, for each 2 mmol/l (36 mg/dl) higher admission glucose, the probability of severe progression significantly increased by about 15% independently from any other clinical-biochemical variable. The association between hyperglycemia and COVID-19 severity was significantly stronger for patients with newly-diagnosed diabetes than for those with pre-existing diabetes. Our finding is consistent with prior literature indicating that, in patients with acute medical conditions, newlydetected diabetes is a powerful predictor of poor outcomes [bib_ref] Diabetes known or newly detected, but not impaired glucose regulation, has a..., Lenzen [/bib_ref] [bib_ref] Hyperglycemia: an independent marker of inhospital mortality in patients with undiagnosed diabetes, Umpierrez [/bib_ref]. This may be at least in part driven by the fact that known diabetes is often associated with manifest organ damage that can be accounted for clinically and statistically [bib_ref] Prognostic significance of silent myocardial infarction in newly diagnosed type 2 diabetes..., Davis [/bib_ref]. Vice-versa, patients who are unaware of their diabetes status are most of the times unaware of ongoing organ damage too, as their treating physicians are. In addition, such occult organ damage cannot be accounted for in statistical adjustment. Yet, our data argue that newly-diagnosed diabetes was predictive of COVID-19 severity not only because of a possible masked multi-organ frailty, but also by the strong link between glucose levels and the outcome. We performed an analysis to evaluate which of the clinical-laboratory parameters of disease severity were mostly responsible for mediating the effect of hyperglycemia on the outcome. Top results included respiratory parameters and white blood cell count, whereas other markers of inflammation and tissue damage were not among top mediators, suggesting that hyperglycemia drove a worse progression of respiratory failure. Indeed, PaO 2 /FiO 2 directly reflects respiratory failure and drives ICU admittance or mechanical ventilation.
Diabetes and hyperglycemia were previously found to cause a form of pulmonary disease known as ''diabetic lung", featuring changes in lung volumes and diffusion capacity [bib_ref] Diabetic lung, an underrated complication from restrictive functional pattern to pulmonary hypertension, Fuso [/bib_ref]. Speculatively, patients with admission hyperglycemia upon hospitalization for COVID-19 might have a poorer outcome because of an underlying subclinical pulmonary remodeling [bib_ref] The diabetic lung: an easy target for SARS-CoV-2?, Caruso [/bib_ref]. The diabetic lung is supposed to be driven at least in part by obesity and metabolic syndrome [bib_ref] The metabolic syndrome, diabetes and lung dysfunction, Tiengo [/bib_ref] , but systemic inflammation and platelet dysfunction have been implicated in hyperglycemic pulmonary microangiopathy [bib_ref] Diabetic microvascular disease and pulmonary fibrosis: the contribution of platelets and systemic..., Jagadapillai [/bib_ref]. Since inflammatory overactivation and coagulopathy are major fea-tures of severe COVID-19, we speculate that hyperglycemia can directly accelerate disease course.
The study has limitations that do not allow us to exclude alternative explanations. First, some clinical variables were not collected for all patients, due to the setting where COVID-19 patients were followed, i.e. strictly isolated wards with limited contacts with health personnel. Absence of data on body mass index (BMI) prevented us from adequately considering the confounding role of obesity [bib_ref] Predicting mortality due to SARS-CoV-2: A mechanistic score relating obesity and diabetes..., Bello-Chavolla [/bib_ref] [bib_ref] Obesity is associated with worse outcomes in COVID-19: analysis of early data..., Hajifathalian [/bib_ref]. In addition, HbA1c was not available for all patients, potentially biasing the detection of pre-existing hyperglycemia. Although we were able to control for the use of preadmission use of glucocorticoids, in most cases it was impossible to judge the duration of hyperglycemia prior to hospitalization for COVID-19 in patients without known diabetes. Therefore, whether these patients had undiagnosed diabetes or stress hyperglycemia due to the cytokine storm remains unclear. Despite our attempts to control for major biases, we cannot rule out that residual confounding was driving a spurious non-causal association between hyperglycemia and severe COVID-19. For example, a more severe degree of inflammatory activation before hospitalization could drive both stress hyperglycemia and a subsequent severe disease course, even if hyperglycemia exerted no direct role on disease progression. In other terms, it is impossible to rule out that hyperglycemia is simply a biomarker of a more severe disease [bib_ref] Hyperglycemia: an independent marker of inhospital mortality in patients with undiagnosed diabetes, Umpierrez [/bib_ref].
Nonetheless, our study has notable strengths, including the detailed clinical characterization of patients with inclusion of several biochemical inflammatory markers, and the rigorous statistical approach to bias. Furthermore, the mediation analysis allows speculating on the processes that drove poor outcome of COVID-19 patients presenting with admission hyperglycemia. .
Finally, we wish to underline that, while this manuscript was under preparation, a study conducted on 453 patients hospitalized for COVID-19 at the Union Hospital in Wuhan (China) found that newly diagnosed diabetes was associated with a higher risk of mortality than known diabetes [bib_ref] Newly diagnosed diabetes is associated with a higher risk of mortality than..., Li [/bib_ref]. Interestingly, patients without known diabetes but presenting with hyperglycemia had the worst outcome. The similarities with our study are striking, providing evidence that COVID-19 presents with the same clinical features in distant countries and different healthcare models. This also markedly reinforces the clinical message that hyperglycemia is a strong prognostic factor for adverse outcomes during COVID-19.
## Data availability
Original data are available from the corresponding author at a reasonable request.
# Contribution statement
# Funding
The study was supported by the University of Padova Department of Medicine with a grant from Fondazione CA.RI.PA.RO (COVIDIMED project), Italy.
## Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
[fig] Figure 2 -: Correlation between admittance glucose levels and severity clinical variables. Clinical variables of COVID-19 severity are divided into those related to hemodynamics, respiratory, inflammatory, hematologic and tissue damage biomarkers. For each correlation plot, we show individual data points, the regression line with shadowed 95% C.I. between dashed lines, as well as the Pearson's r correlation coefficient. *p < 0.05. When distribution of the dependent variable was highly skewed, the Y scale was log transformed. [/fig]
[fig] Figure 3 -: Mediators of the effects of hyperglycemia on COVID-19 outcomes. For each candidate mediator, the forest plot shows the association with the primary COVID-19 severity outcome (per each higher or lower SD), along with a histogram of their mediation effect on the association between glucose levels and COVID-19 severity. Mediation is expressed as percentage of the glucose effect on the primary outcome. Further details on the methods are given in the text, whereas full data are presented in [/fig]
[table] Table 2 -: Hospital treatments and outcomes according to diabetes status. In patients without diabetes and in those with diabetes (pre-existing and newly-diagnosed altogether), we show the worst value of clinical-laboratory parameters, as well as in-hospital treatments and outcomes. [/table]
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Gut Micro- and Mycobiota in Preeclampsia: Bacterial Composition Differences Suggest Role in Pathophysiology
Preeclampsia is a severe pregnancy-related inflammatory disease without an effective treatment. The pathophysiology remains partly unknown. However, an increased inflammatory response and oxidative stress are part of the maternal systemic reaction. Recent data have suggested that dysbiosis of the gut microbiome plays a role in preeclampsia as well as other inflammatory diseases. However, dysbiosis in preeclampsia has not been studied in a Scandinavian population. Furthermore, although the fungal flora may also have anti-inflammatory properties, it has never been studied in preeclampsia. We included 25 preeclamptic and 29 healthy third-trimester women for the ITS and 16S sequencing of fungal and bacterial microbiota, respectively. Calprotectin was measured to assess systemic and intestinal inflammatory responses. The fungal diversity differed with BMI and gestational length, suggesting a link between fungi and the immune changes seen in pregnancy. An LEfSe analysis showed 18 significantly differentially abundant bacterial taxa in PE, including enriched Bacteroidetes and depleted Verrucomicrobia and Syntergistota at the phylum level and depleted Akkermansia at the genus level, suggesting a role in the pathophysiology of PE.
# Introduction
Preeclampsia (PE) is a leading cause of maternal and fetal morbidity and mortality worldwide. To date, the only cure is to deliver the baby and thereby the placenta.
PE is a pregnancy-specific condition characterized by gestational hypertension in combination with proteinuria and/or other manifestations of maternal organ dysfunction, including fetal growth restriction, arising after 20 weeks of pregnancy [bib_ref] The hypertensive disorders of pregnancy: ISSHP classification, diagnosis & management recommendations for..., Brown [/bib_ref]. Globally, 3-7% of all pregnancies are affected, which constitutes a total of 8.5 million women every year. A range of short-and long-term complications can follow for both the mother and the child, including an increased long-term risk of cardiovascular disease [bib_ref] Preeclampsia: Pathophysiology, Challenges, and Perspectives, Rana [/bib_ref] [bib_ref] Pre-eclampsia and risk of cardiovascular disease and cancer in later life: Systematic..., Bellamy [/bib_ref]. Risk factors for developing PE include nulliparity, advanced age of the mother (≥40 years), obesity, diabetes, hypertension, renal disease, the presence of antiphospholipid antibodies, in vitro fertilization, and a family history of PE [bib_ref] The hypertensive disorders of pregnancy: ISSHP classification, diagnosis & management recommendations for..., Brown [/bib_ref]. Many of these risk factors overlap with risk factors for cardiovascular disease.
The underlying etiology behind PE is still largely unknown. However, placental stress is central to the pathophysiology, with different etiologies in early-onset (<34 gestational weeks) and late-onset PE (≥34 gestational weeks) [bib_ref] Syncytiotrophoblast stress in preeclampsia: The convergence point for multiple pathways, Redman [/bib_ref]. Early-onset PE is mainly caused by defective placentation due to a shallow migration of cytotrophoblasts into decidual spiral arteries, resulting in defective vascular remodeling [bib_ref] Syncytiotrophoblast stress in preeclampsia: The convergence point for multiple pathways, Redman [/bib_ref] [bib_ref] Placental bed research: I. The placental bed: From spiral arteries remodeling to..., Brosens [/bib_ref]. This gives rise to hypoperfusion, ischemia, oxidative stress, and tissue damage in the placenta. In late-onset PE, placental stress is likely caused by a combination of senescence of the placenta and a maternal genetic predisposition to metabolic and cardiovascular disease [bib_ref] Syncytiotrophoblast stress in preeclampsia: The convergence point for multiple pathways, Redman [/bib_ref]. In stage two, characterized by the maternal syndrome, the leakage of debris and placenta-derived vesicles from the fetoplacental unit to the maternal circulation contributes to the systemic inflammation, endothelial damage, and dysfunction that are central in PE [bib_ref] Syncytiotrophoblast stress in preeclampsia: The convergence point for multiple pathways, Redman [/bib_ref] [bib_ref] The myometrial junctional zone spiral arteries in normal and abnormal pregnancies: A..., Brosens [/bib_ref] [bib_ref] The roles of free iron, heme, haemoglobin, and the scavenger proteins haemopexin..., Erlandsson [/bib_ref] [bib_ref] The Two Stage Model of Preeclampsia: Variations on the Theme, Roberts [/bib_ref].
There are increases in proinflammatory cytokines as well as calprotectin, an inflammatory marker commonly used in the clinic, in the maternal circulation in PE [bib_ref] Association of pro-and anti-inflammatory cytokines in preeclampsia, Aggarwal [/bib_ref] [bib_ref] Calprotectin in pregnancy and pregnancy-associated diseases: A systematic review and prospective cohort..., Rezniczek [/bib_ref]. Calprotectin is a heterodimer protein that is mainly expressed in myeloid cells, functioning as a promotor and amplifier of inflammation in various diseases [bib_ref] The endogenous Toll-like receptor 4 agonist S100A8/S100A9 (calprotectin) as innate amplifier of..., Ehrchen [/bib_ref]. It binds to pattern recognition receptor toll-like receptor 4 (TLR4) on the cell surfaces of monocytes, activating the innate immune system [bib_ref] Mrp8 and Mrp14 are endogenous activators of Toll-like receptor 4, promoting lethal,..., Vogl [/bib_ref]. TLR4 also recognizes lipopolysaccharide (LPS) from the outer surface membranes of Gram-negative bacteria. A significant increase in serum calprotectin levels has been described in the third trimester of PE pregnancies [bib_ref] An evaluation of calprotectin as serum marker of preeclampsia: A systematic review..., Pergialiotis [/bib_ref].
There is increasing evidence that dysbiosis of the gut microbiome and increased intestinal permeability are involved in several diseases that often involve low-grade inflammation, such as obesity, diabetes, and hypertension [bib_ref] Metabolic endotoxemia initiates obesity and insulin resistance, Cani [/bib_ref] [bib_ref] Involvement of gut microbiota in the development of low-grade inflammation and type..., Cani [/bib_ref] [bib_ref] Linking Gut Microbiota and Inflammation to Obesity and Insulin Resistance, Saad [/bib_ref] [bib_ref] Gut microbiota in hypertension, Jose [/bib_ref]. The gut microbiome plays important roles in host metabolism, immune responses, intestinal barrier function, and intestinal permeability [bib_ref] Intestinal microbiota, diet and health, Power [/bib_ref] [bib_ref] The gut flora as a forgotten organ, O'hara [/bib_ref]. Different bacterial strains can convey different effects in the host, for example, through differences in short-chain fatty acid (SCFA) production or the amount of LPS on the cell surface [bib_ref] Linking Gut Microbiota and Inflammation to Obesity and Insulin Resistance, Saad [/bib_ref] [bib_ref] Short-chain fatty acids accompanying changes in the gut microbiome contribute to the..., Chang [/bib_ref] [bib_ref] Effects of Butyrate on Intestinal Barrier Function in a Caco-2 Cell Monolayer..., Peng [/bib_ref] [bib_ref] Microbiota and SCFA in Lean and Overweight Healthy Subjects, Schwiertz [/bib_ref]. It is thought that LPS translocation over an impaired gut-blood barrier is a contributor to systemic inflammation, with several other effects to follow, such as increased insulin resistance [bib_ref] Metabolic endotoxemia initiates obesity and insulin resistance, Cani [/bib_ref] [bib_ref] Involvement of gut microbiota in the development of low-grade inflammation and type..., Cani [/bib_ref] [bib_ref] Linking Gut Microbiota and Inflammation to Obesity and Insulin Resistance, Saad [/bib_ref]. The use of probiotics has often shown beneficial effects on these parameters [bib_ref] Intestinal microbiota, diet and health, Power [/bib_ref] [bib_ref] Probiotic Lactobacillus Species Strengthen Intestinal Barrier Function and Tight Junction Integrity in..., Blackwood [/bib_ref].
The fungal component of the gut flora, the mycobiome, is far less studied. Fungi make up around 0.1% of the gut microbiome [bib_ref] A human gut microbial gene catalogue established by metagenomic sequencing, Qin [/bib_ref] , but as a fungal cell is >100 times larger than a bacterial cell, fungi still make up a substantial part of the microbiome biomass [bib_ref] The mycobiota: Interactions between commensal fungi and the host immune system, Underhill [/bib_ref]. The gut mycobiome is known to play a role in modulating immune responses both locally and systemically in the host [bib_ref] Gut Mycobiota in Immunity and Inflammatory Disease, Li [/bib_ref]. Decreased mycobiome family biodiversity has been shown in obesity, and mycobiome dysbiosis has been shown in IBD [bib_ref] Fungi and inflammatory bowel diseases: Alterations of composition and diversity, Ott [/bib_ref] [bib_ref] Dysbiosis of Gut Fungal Microbiota is Associated With Mucosal Inflammation in Crohn's..., Li [/bib_ref]. However, to the best of our knowledge, the gut mycobiota has not been studied in PE.
During a normal pregnancy, a shift in the gut microbiome composition is seen, including a decrease in diversity and an increase in the abundance of the phyla Proteobacteria and Actinobacteria [bib_ref] Host Remodeling of the Gut Microbiome and Metabolic Changes during Pregnancy, Koren [/bib_ref]. Proteobacteria are often associated with inflammation in non-pregnancy but might play a more beneficial role in pregnancy [bib_ref] IBD-what role do Proteobacteria play?, Mukhopadhya [/bib_ref]. These changes are accompanied by metabolic changes that are beneficial and necessary for a normal pregnancy, such as increased fat accumulation in the first trimester and increased insulin resistance in the third trimester [bib_ref] Cellular Mechanisms for Insulin Resistance in Normal Pregnancy and Gestational Diabetes, Barbour [/bib_ref]. However, in obese pregnant women and pregnancies complicated by PE, studies have shown differential gut microbiome compositions [bib_ref] Short-chain fatty acids accompanying changes in the gut microbiome contribute to the..., Chang [/bib_ref] [bib_ref] Gut Microbiota Richness and Composition and Dietary Intake of Overweight Pregnant Women..., Mokkala [/bib_ref] [bib_ref] Pre-Eclampsia: Microbiota possibly playing a role, Ahmadian [/bib_ref] [bib_ref] Remodeling of the gut microbiota and structural shifts in Preeclampsia patients in..., Liu [/bib_ref] [bib_ref] Early-Onset Preeclampsia Is Associated With Gut Microbial Alterations in Antepartum and Postpartum..., Lv [/bib_ref] [bib_ref] Gut Microbiota Dysbiosis and Increased Plasma LPS and TMAO Levels in Patients..., Wang [/bib_ref] [bib_ref] Gut dysbiosis induces the development of pre-eclampsia through bacterial translocation, Chen [/bib_ref]. One study has shown a causal relationship between dysbiosis and PE through bacterial translocation in a mouse model that received fecal microbiota transplantation (FMT) from PE patients [bib_ref] Gut dysbiosis induces the development of pre-eclampsia through bacterial translocation, Chen [/bib_ref]. Other studies indicated that gut dysbiosis leads to blood pressure elevation and PE development, mainly through the effects of dysregulated plasminogen activator inhibitor-1 (PAI-1), butyrate, and other SCFAs [bib_ref] Gut dysbiosis induces the development of pre-eclampsia through bacterial translocation, Chen [/bib_ref] [bib_ref] Increased Systolic and Diastolic Blood Pressure Is Associated with Altered Gut Microbiota..., Gomez-Arango [/bib_ref] [bib_ref] Gut Dysbiosis Promotes Preeclampsia by Regulating Macrophages and Trophoblasts, Jin [/bib_ref]. A recent study showed that a lower abundance of the butyrate-producing genus Coprococcus, as well as lower serum butyrate levels, can already be detected at 28 gestational weeks in obese women who later develop late-onset PE. The MoBa study in Norway has shown that the consumption of milk-based probiotics lowers the risk of developing PE [bib_ref] Intake of Probiotic Food and Risk of Preeclampsia in Primiparous Women: The..., Brantsaeter [/bib_ref] [bib_ref] Timing of probiotic milk consumption during pregnancy and effects on the incidence..., Nordqvist [/bib_ref].
Although studies have shown an altered gut flora in PE patients, the number of studies is limited [bib_ref] Remodeling of the gut microbiota and structural shifts in Preeclampsia patients in..., Liu [/bib_ref] [bib_ref] Early-Onset Preeclampsia Is Associated With Gut Microbial Alterations in Antepartum and Postpartum..., Lv [/bib_ref] [bib_ref] Gut Microbiota Dysbiosis and Increased Plasma LPS and TMAO Levels in Patients..., Wang [/bib_ref] [bib_ref] Gut dysbiosis induces the development of pre-eclampsia through bacterial translocation, Chen [/bib_ref] [bib_ref] Gut Dysbiosis Promotes Preeclampsia by Regulating Macrophages and Trophoblasts, Jin [/bib_ref] [bib_ref] Decrease in abundance of bacteria of the genus Bifidobacterium in gut microbiota..., Miao [/bib_ref] , and there might be differences among populations due to environmental differences. Studies in Scandinavian populations are scarce. Furthermore, the mycobiome has not yet been studied in PE or in normal pregnancy. Another aspect that has been overlooked in studies of the gut microbiome in PE is the presence of symptoms from the gastrointestinal tract, such as loose/watery stool and local inflammation in the intestine.
Studies on possible roles of the gut microbiome in PE contribute to new knowledge regarding the pathophysiology of a very common complication of pregnancy. They also provide potential new targets for prediction and treatment. Our hypothesis is that the damaged endothelium in the vascular bed causes an impaired gut-blood barrier, indirectly affecting the gut microbiota, and that these changes can cause or aggravate the inflammatory response both systemically and locally in the gut.
The purpose of this study was therefore to evaluate differences in both the bacterial and fungal components of the gut microbiome in women with PE compared to gestational age-matched controls in southern Sweden. This was performed using 16S and ITS rRNA gene sequencing for bacteria and fungi, respectively. In addition, the calprotectin levels were evaluated in maternal feces and blood as a marker of inflammation, and symptoms in the gastrointestinal tract were evaluated.
# Materials and methods
# Ethics statement
The study was approved by the Regional Ethics Committee at Lund University, Sweden (2017/419). All subjects gave their written, informed consent before entering the study. All samples were coded before analysis.
## Definitions
PE was defined according to the ISSHP 2018 diagnostic criteria [bib_ref] The hypertensive disorders of pregnancy: ISSHP classification, diagnosis & management recommendations for..., Brown [/bib_ref] : de novo hypertension (systolic blood pressure ≥140 mmHg or diastolic blood pressure ≥90 mmHg) manifesting after gestational week 20, combined with proteinuria or signs of organ dysfunction (thrombocytopenia, impaired liver function, progressive renal insufficiency, pulmonary edema, or new-onset cerebral of visual disturbances). Severe PE was defined as a systolic blood pressure ≥160 mmHg, a diastolic blood pressure ≥110 mmHg, or the prevalence of the organ dysfunctions mentioned above. Early-onset PE was defined as PE manifesting before 34 gestational weeks, and late-onset PE manifested after 34 gestational weeks.
## Study cohort and questionnaire
Patients in the third trimester with newly diagnosed PE or normotensive control pregnancies were recruited for the study. Recruitment took place between 2017 and 2020 at delivery wards at Skåne University Hospital and at selected antenatal care centers in Malmö and Lund, Scania, Sweden. The exclusion criteria were type 1 or 2 diabetes, gestational diabetes in the current pregnancy, IBD, celiac disease, BMI > 30 before pregnancy, and treatment with antibiotics within the past 2 weeks.
A questionnaire on diet regarding the intake of probiotics and meat, medications, obstetric and medical history, and smoking habits as well as symptoms related to PE or the gastrointestinal tract was filled in by the patients. The questionnaire was designed to give an overview of factors that could be related to variations in the studied parameters and was partly based on questionnaires for the screening and monitoring of IBD and irritable bowel syndrome (IBS) patients. Demographic data and the results of a routine oral glucose tolerance test (OGTT) performed during gestational week 28 were acquired from medical records.
## Sample collection
Blood samples were drawn from the antecubital vein, and feces samples were collected in sterile 20 mL tubes at inclusion. The samples were immediately stored in a fridge for a maximum of 48 h. The blood samples were centrifuged, and plasma was stored at −80 - C until analysis. The feces samples were stored at −80 - C until further analysis.
## Protein extraction and calprotectin elisa
The calprotectin concentration in feces and plasma was determined using a twosite sandwich ELISA technique (Immundiagnostik AG, Bensheim, Germany). A total of 15 mg of feces from each patient sample was used for protein extraction according to manufacturer's protocol. The absorbance was measured at 450 nm, and a curve was fitted to standard samples using a 4-parameter algorithm. Calprotectin concentrations were calculated from the resulting equations.
## Reference values
Feces calprotectin >50 mg/kg is considered borderline-positive, and >100 mg/kg is considered positive, according to manufacturer. Factors that can increase the levels of fecal calprotectin include the use of NSAIDs (non-steroidal anti-inflammatory drugs), intercurrent gastrointestinal infection, and malignancy. The manufacturer gave no reference value for plasma calprotectin since levels can vary depending on the preanalytical conditions. Variations in preanalytical conditions were kept to a minimum in our study.
## Dna extraction, 16s rrna, and its1 gene amplicon sequencing
Starting with the 54 enrolled women (29 C and 25 PE), 46 fecal samples (26 C and 20 PE) were collected and underwent total genomic DNA extraction. The taxonomic structure of the gut microbiome can be defined using DNA amplification and the sequencing of hypervariable regions in highly conserved sequences of the genome. For bacteria, hypervariable regions of the highly conserved 16S rRNA gene are used for this purpose. The standard marker used in fungal taxonomy is the internal transcribed spacer (ITS) region of the rRNA gene. The generated sequences are clustered to generate operational taxonomic units (OTUs), which can be compared with reference libraries to define the taxonomic structure at the species level.
Total gDNA was extracted from the fecal samples using the DNeasy PowerSoil Pro kit (QIAGEN, Venlo, The Netherlands) and subsequently quantified with fluorometric quantification using the Invitrogen Qubit Fluorometer assay (Thermofisher, Waltham, MA, USA). Specific primers were used to target the hypervariable V3-V4 regions of the 16S gene (341f: 5 -CCTACGGGNGGCWGCAG-3 and 805r: 5 -GACTACNVGGGTWTCTAATCC-3 ) [bib_ref] Development of a Prokaryotic Universal Primer for Simultaneous Analysis of Bacteria and..., Takahashi [/bib_ref] and the ITS1 region (ITS1f: 5 -CTTGGTCATTTAGAGGAAGTAA-3 and ITS2r: 5 -GCTGCGTTCTTCATCGATGC-3 ) [bib_ref] Development of a Prokaryotic Universal Primer for Simultaneous Analysis of Bacteria and..., Takahashi [/bib_ref] , respectively, for the bacterial and fungal microbiota, and were fitted with overhang Illumina adapters. Amplicon library preparation was conducted according to Illumina protocols. Paired-end sequencing was carried out using a 600-cycle MiSeq™ Reagent Kit v3 and an Illumina MiSeq system (Illumina, San Diego, CA, USA) at the Biology Department of Florence University, Italy.
## Sequence data processing
The DADA2 pipeline (version 1.14.1) was used to reconstruct amplicon sequence variants (ASVs) from the fastq files. Sequence variant reconstruction and statistical analyses were performed in the R environment (version 3.4.3). Amplification primers were detected and removed from the Illumina raw data using cutadapt (version 1.15). Low-quality reads were discarded using the filterAndTrim function. A minimum length of 50 bp was set to avoid technical constructs that could be created during the amplification step. The DADA2 function was used to perform denoising after error rate remodeling (learnErrors function). Forward and reverse reads were merged using the mergePairs function. Chimeric sequences were removed using the removeBimeraDenovo function. A taxonomic classification was assigned to the sequence variants using DECIPHER Bioconductor package version 2.14.0. For ITS sequencing, the reference database used was the RDP classifier Warcup ITS training dataset. The ASV table output sequences were analyzed using the phyloseq R package.
## Statistical analyses
SPSS Statistics 27 (IBM) was used for Mann-Whitney U-tests to compare patient demographics, the levels of calprotectin in plasma and feces, and oral glucose tolerance test (OGTT) values between PE and normotensive groups. Kruskal-Wallis tests were performed to compare questionnaire answers regarding smoking, gastrointestinal tract symptoms, diet, and probiotics consumption.
For microbiota analyses, R software and the vegan package (version 2.5-6) were used. Alpha diversity was analyzed using a one-way analysis of variance (ANOVA), and beta diversity was analyzed using the Bray-Curtis dissimilarity index using the vegdist function. Differences in sample composition were analyzed using a principal coordinate analysis (PCoA) based on vegdist among all samples using the cmdscale R function. The effects of diet and PE on variances in microbiota composition among samples were studied using a permutational multivariate analysis of variance (PERMANOVA, adonis2 function of the vegan package with 1000 permutations). The effects of gestational age and BMI on variances in microbiota composition were analyzed using an environmental fitting using the R function envfit with 1000 permutations. A t-test was performed to investigate the difference between two distinguishable groups in the analysis. DESeq2 was used to estimate fold changes in the gut mycobiota between normotensive pregnancy and PE. Differential abundances in the bacterial microbiota between groups were analyzed using a linear discriminant analysis effect size (LEfSe) analysis, with LDA scores = 3.
Results with p values < 0.05 were considered significant.
# Results
## Patient characteristics and questionnaire
A total of 54 women were included in the study. In total, five individuals from the PE group and three individuals from the control group failed to leave feces samples, and blood samples were not collected from one individual from the control group. One individual from the PE group was excluded from calculations regarding patient characteristics since they had a duplex pregnancy. The final inclusions were 25 PE patients (20 feces samples and 25 blood samples) and 29 women with normotensive pregnancies, designated as controls (26 feces samples and 29 blood samples).
The patient demographics for singleton pregnancies are summarized in . The groups were similar with regards to age, pre-pregnancy BMI, gestational age at inclusion, and fetal sex ratio. There were more nulliparous women in the PE group. Gestational age at birth and birth weight were significantly lower in the PE group. The control group and PE group had similar smoking habits and dietary habits regarding the consumption of meat; other foods of animal origin; and probiotic foods such as fermented milk, yoghurt, kombucha, miso, tempeh, and other foods or supplements containing probiotics. . Patient demographics, questionnaire answers (smoking, diet, and probiotic food consumption), and OGTT results for singleton pregnancies included in the present study 1 . The results from the questionnaire regarding gastrointestinal symptoms showed that patients with PE had significantly more frequent bowel movements and a tendency towards more loose/watery stool than the controls (p = 0.024 and p = 0.059, respectively) .
## Parameter
## Controls (c) preeclampsia (pe) p-value
## Oral glucose tolerance test at gestational week 28
The Mann-Whitney U-test showed a significant difference in the 2 h glucose levels during the oral glucose tolerance test (OGTT) at 28 weeks, with lower levels in the group that later developed PE (p = 0.030). For the controls (n = 28) the median blood glucose level was 6.65 mmol/L (IQR: 6.13-7.15 mmol/L), while the median level in the PE group (n = 21) was 6.10 mmol/L (IQR: 5.10-6.70 mmol/L) .
## Calprotectin levels in plasma and feces
A Mann-Whitney U-test showed significantly higher plasma calprotectin levels in the PE group (N = 25, median 3764 µg/L, IQR 2897-13,639 µg/L) compared to controls (N = 28, median 1819 µg/L, IQR 1176-2612 µg/L) (p < 0.001), with high variation between subjects [fig_ref] Figure 1: Plasma [/fig_ref]. The difference remained significant (p < 0.001) after excluding outliers, defined as values greater than Q3 + 1.5*IQR for each group, which excluded two PE patients and three controls [fig_ref] Figure 1: Plasma [/fig_ref]. There was no significant difference in feces calprotectin levels
## Micro-and mycobiomes based on 16s and its rrna gene sequencing
Using ITS1 and 16S rRNA gene amplicon sequencing, the fungal and bacterial communities were profiled, respectively, for 43 (24 C and 19 PE) and 42 samples. Samples that generated insufficient library concentrations due to amplification problems were excluded (one PE patient and two controls for ITS1 amplification and three PE patients and one control for 16S rRNA amplification). The average number of reads obtained for each sample from MiSeq ITS sequencing was 157，543. After the Dada2 pipeline was used to construct ASVs, the average number of reads for each sample was 12， 769. After removing adapters, filtering, and removing unknown ASVs, 345 known ASVs remained, representing around 10% of the total number of reads. The reduction in retained reads after all the ASV detection steps of the pipeline was approximately 90%. This result is explained by the fact that most fungal diversity is unknown, and ITS sequences in the International Nucleotide Sequence Database (INSD: GenBank, EMBL, and DDBJ) revealed that this region is not equally variable in all groups of fungi. The 16s rRNA sequencing generated reads with a quality compatible with downstream analysis for 39 samples (24 C and 15 PE). The ITS1 sequencing was successful for all 43 amplicon libraries. All metagenome sequences were deposited in the European Nucleotide Archive under Bioproject PRJEB59212.
## Diversity indices
Between-sample dissimilarities (i.e., beta diversity) were evaluated using a classical multidimensional scaling (MDS) of a data matrix, also known as a principal coordinate analysis (PCoA) on a quantitative index (Bray-Curtis). The beta diversity across fungal and bacterial communities in control and PE pregnancies, visualized in PCoA plots, did
## Micro-and mycobiomes based on 16s and its rrna gene sequencing
Using ITS1 and 16S rRNA gene amplicon sequencing, the fungal and bacterial communities were profiled, respectively, for 43 (24 C and 19 PE) and 42 (25 C and 17 PE) samples. Samples that generated insufficient library concentrations due to amplification problems were excluded (one PE patient and two controls for ITS1 amplification and three PE patients and one control for 16S rRNA amplification). The average number of reads obtained for each sample from MiSeq ITS sequencing was 157,543. After the Dada2 pipeline was used to construct ASVs, the average number of reads for each sample was 12,769. After removing adapters, filtering, and removing unknown ASVs, 345 known ASVs remained, representing around 10% of the total number of reads. The reduction in retained reads after all the ASV detection steps of the pipeline was approximately 90%. This result is explained by the fact that most fungal diversity is unknown, and ITS sequences in the International Nucleotide Sequence Database (INSD: GenBank, EMBL, and DDBJ) revealed that this region is not equally variable in all groups of fungi. The 16s rRNA sequencing generated reads with a quality compatible with downstream analysis for 39 samples (24 C and 15 PE). The ITS1 sequencing was successful for all 43 amplicon libraries. All metagenome sequences were deposited in the European Nucleotide Archive under Bioproject PRJEB59212.
## Diversity indices
Between-sample dissimilarities (i.e., beta diversity) were evaluated using a classical multidimensional scaling (MDS) of a data matrix, also known as a principal coordinate analysis (PCoA) on a quantitative index (Bray-Curtis). The beta diversity across fungal and bacterial communities in control and PE pregnancies, visualized in PCoA plots, did not show any significant differences between the groups [fig_ref] Figure 2: Diversity indices for fungal and bacterial microbiome composition in control and PE... [/fig_ref]. The alpha diversity in the bacterial and fungal microbiomes, according to the Chao1 and Shannon diversity indices, did not differ between the groups. [fig_ref] Figure 2: Diversity indices for fungal and bacterial microbiome composition in control and PE... [/fig_ref]. This group could not be distinguished in the 16S analysis. This group was renamed BMI+ since it was arranged in a position compatible with the direction of the BMI vector [fig_ref] Figure 2: Diversity indices for fungal and bacterial microbiome composition in control and PE... [/fig_ref]. The remaining samples were included in a second group, renamed BMI-, where the samples were arranged opposite the BMI vector direction. A T-test showed that the BMI+ group had a significantly higher pre-pregnancy BMI (mean of 25.65 kg/m 2 compared with 23.36 kg/m 2 , p = 0.0361) as well as a lower gestational age at inclusion (mean of 220 days compared with 246 days, p = 0.0411) [fig_ref] Figure 2: Diversity indices for fungal and bacterial microbiome composition in control and PE... [/fig_ref]. An environmental fitting analysis showed that gestational age was positively correlated with PCoA1 and negatively correlated with PCoA2, with an R-squared value of 0.326 (p = 0.002). BMI was negatively correlated with PCoA1, but the R-squared value was only 0.098 (p = 0.192).
The R-squared values obtained with a PERMANOVA support that neither diet nor PE explain the variations in the bacterial microbiome composition (R-squared = 0.050 and 0.031, respectively) or mycobiome composition (R-squared = 0.036 and 0.018, respectively). [fig_ref] Figure 2: Diversity indices for fungal and bacterial microbiome composition in control and PE... [/fig_ref]. This group could not be distinguished in the 16S analysis. This group was renamed BMI+ since it was arranged in a position compatible with the direction of the BMI vector [fig_ref] Figure 2: Diversity indices for fungal and bacterial microbiome composition in control and PE... [/fig_ref]. The remaining samples were included in a second group, renamed BMI-, where the samples were arranged opposite the BMI vector direction. A t-test showed that the BMI+ group had a significantly higher pre-pregnancy BMI (mean of 25.65 kg/m 2 compared with 23.36 kg/m 2 , p = 0.0361) as well as a lower gestational age at inclusion (mean of 220 days compared with 246 days, p = 0.0411) [fig_ref] Figure 2: Diversity indices for fungal and bacterial microbiome composition in control and PE... [/fig_ref]. An environmental fitting analysis showed that gestational age was positively correlated with PCoA1 and negatively correlated with PCoA2, with an R-squared value of 0.326 (p = 0.002). BMI was negatively correlated with PCoA1, but the R-squared value was only 0.098 (p = 0.192).
The R-squared values obtained with a PERMANOVA support that neither diet nor PE explain the variations in the bacterial microbiome composition (R-squared = 0.050 and 0.031, respectively) or mycobiome composition (R-squared = 0.036 and 0.018, respectively).
## Gut bacterial microbiome composition
At the phylum level, we saw that Firmicutes were dominant in most patients, followed by Bacteroidetes [fig_ref] Figure 3: Stacked bar plots of the relative abundance with the standard deviation of... [/fig_ref]. The relative abundance of Firmicutes was lower in PE, especially the class Clostridia, although this was not significant (p > 0.05) [fig_ref] Figure 3: Stacked bar plots of the relative abundance with the standard deviation of... [/fig_ref]. The differential abundances in the bacterial microbiota between groups were analyzed using a linear discriminant analysis effect size (LEfSe) analysis, and the significantly differentially abundant taxa are summarized in [fig_ref] Figure 4: Linear discriminant analysis effect size [/fig_ref].
## Gut bacterial microbiome composition
At the phylum level, we saw that Firmicutes were dominant in most patient lowed by Bacteroidetes [fig_ref] Figure 3: Stacked bar plots of the relative abundance with the standard deviation of... [/fig_ref]. The relative abundance of Firmicutes was lower especially the class Clostridia, although this was not significant (p > 0.05) (Figure 3b differential abundances in the bacterial microbiota between groups were analyzed a linear discriminant analysis effect size (LEfSe) analysis, and the significantly dif tially abundant taxa are summarized in [fig_ref] Figure 4: Linear discriminant analysis effect size [/fig_ref]. This large effect size with a high p-value (>0.05%) is due to the high variability of the Clostridia class in the PE samples. Random sampling error is more likely to produce substantial differences between groups, even when no effect exists in the population.
Verrucomicrobia were present in most patients at varying, mostly low levels, while Desulfobacteria and Synergistia were present only at low levels. Proteobacteria also displayed a very low relative abundance in all patients except for one PE patient, where they constituted around 35%. The relative abundance of the class Actinobacteria was lower in the PE group [fig_ref] Figure 3: Stacked bar plots of the relative abundance with the standard deviation of... [/fig_ref] , being present in only one PE patient but in all controls, although the difference was not significant.
An LEfSe analysis showed 18 significantly differentially abundant taxa in PE [fig_ref] Figure 4: Linear discriminant analysis effect size [/fig_ref]. At the phylum level, Bacteroidetes were enriched and Verrucomicrobia and Synergistia were depleted. At the class level, Bacteroidia were enriched and Coriobacteriia, Synergistia, and Verrucomicrobiae were depleted. At the order level, Bacteroidales were enriched and Coriobacteriales, Synergistales, and Verrucomicrobiales were depleted. At the family level, Erysipelotrichaceae, Coriobacteriaceae, Synergistaceae, and Akkermansiaceae were depleted. At the genus level, Collinsella, Akkermansia, and Cloacibacillus were depleted. Coriobacteriia, Coriobacteriales, Coriobacteriaceae, and Collinsella belong to the phylum Actinobacteria.
## Gut mycobiome composition
The average number of reads obtained for each sample from MiSeq sequencing was 157.543, and after the Dada2 pipeline was used to construct ASVs, the average number of reads for each sample was 12.769. The reduction in retained reads after all the amplicon sequence variant detection steps of the pipeline was approximately of 90%. This result is explained by the fact that most fungal diversity is unknown, and ITS sequences in the International Nucleotide Sequence Database (INSD: GenBank, EMBL, and DDBJ) revealed that this region is not equally variable in all groups of fungi. For this reason, we did not perform any stratification according to groups but instead show the intestinal mycobiota of all samples, characterizing the mycobiota of pregnant women.
The relative abundances of the most common fungal phyla across all included individuals showed the dominance of Ascomycota, which constituted >50% of the community This large effect size with a high p-value (>0.05%) is due to the high variability of the Clostridia class in the PE samples. Random sampling error is more likely to produce substantial differences between groups, even when no effect exists in the population.
Verrucomicrobia were present in most patients at varying, mostly low levels, while Desulfobacteria and Synergistia were present only at low levels. Proteobacteria also displayed a very low relative abundance in all patients except for one PE patient, where they constituted around 35%. The relative abundance of the class Actinobacteria was lower in the PE group [fig_ref] Figure 3: Stacked bar plots of the relative abundance with the standard deviation of... [/fig_ref] , being present in only one PE patient but in all controls, although the difference was not significant.
An LEfSe analysis showed 18 significantly differentially abundant taxa in PE [fig_ref] Figure 4: Linear discriminant analysis effect size [/fig_ref]. At the phylum level, Bacteroidetes were enriched and Verrucomicrobia and Synergistia were depleted. At the class level, Bacteroidia were enriched and Coriobacteriia, Synergistia, and Verrucomicrobiae were depleted. At the order level, Bacteroidales were enriched and Coriobacteriales, Synergistales, and Verrucomicrobiales were depleted. At the family level, Erysipelotrichaceae, Coriobacteriaceae, Synergistaceae, and Akkermansiaceae were depleted. At the genus level, Collinsella, Akkermansia, and Cloacibacillus were depleted. Coriobacteriia, Coriobacteriales, Coriobacteriaceae, and Collinsella belong to the phylum Actinobacteria.
## Gut mycobiome composition
The average number of reads obtained for each sample from MiSeq sequencing was 157.543, and after the Dada2 pipeline was used to construct ASVs, the average number of reads for each sample was 12.769. The reduction in retained reads after all the amplicon sequence variant detection steps of the pipeline was approximately of 90%. This result is explained by the fact that most fungal diversity is unknown, and ITS sequences in the International Nucleotide Sequence Database (INSD: GenBank, EMBL, and DDBJ) revealed that this region is not equally variable in all groups of fungi. For this reason, we did not perform any stratification according to groups but instead show the intestinal mycobiota of all samples, characterizing the mycobiota of pregnant women.
The relative abundances of the most common fungal phyla across all included individuals showed the dominance of Ascomycota, which constituted >50% of the community in all samples except one, where Basidiomycetes constituted 55% [fig_ref] Figure 5: Bar and stacked bar plots showing relative percentage abundances of mycobiome taxonomic... [/fig_ref]. Basidiomycetes were present, with a mean abundance of 8.4% in 67.4% of the samples. The third detected phylum, Zygomycota, was observed in only two samples (one PE and one C patient), where it constituted minimal traces (<5%).
were present, with a mean abundance of 8.4% in 67.4% of the samples. The third detected phylum, Zygomycota, was observed in only two samples (one PE and one C patient), where it constituted minimal traces (<5%). Subsequently, we tried to reach a deeper taxonomic level of fungal investigation, descending from class to the species level. We reconstructed a stacked area graph using the Subsequently, we tried to reach a deeper taxonomic level of fungal investigation, descending from class to the species level. We reconstructed a stacked area graph using the ggplot2 R package with the classes of fungi and their corresponding relative abundances [fig_ref] Figure 5: Bar and stacked bar plots showing relative percentage abundances of mycobiome taxonomic... [/fig_ref].
[formula] (a) (b) (c) [/formula]
At the class level, the most abundant class, with a mean relative abundance of 50%, was the Saccharomycetes class, followed by the Dothideomycetes and Sordariomycetes classes, present in 70% of the samples at a mean abundance of 14% each, all belonging to the Ascomycota phylum [fig_ref] Figure 5: Bar and stacked bar plots showing relative percentage abundances of mycobiome taxonomic... [/fig_ref]. A graphic examination of the relative abundances at the class level confirmed the results from the diversity analysis, showing no differences in fungal gut microbial profiles between PE and controls. The average abundances of the most abundant fungal species across all samples showed that Candida species were most abundant, reaching a relative abundance of 35% with the species Candida sp VVT_2012. However, other species such as Candida mesorugosa, Candida tropicalis, Candida parapsilosis, and Candida albicans, albeit in a decreasing order of relative abundance, were important colonizers of the microbiomes of pregnant women [fig_ref] Figure 5: Bar and stacked bar plots showing relative percentage abundances of mycobiome taxonomic... [/fig_ref].
The only significant fold change that was calculated between control and PE was for ASV-36 (corresponding to the genus Peniophora), which was less represented in PE patients than in controls.
# Discussion
In this study we report significantly differential abundance in 18 bacterial taxa in PE, including enrichment at the phylum level of Bacteroidetes, and depletion of Verrucomicrobia and Synergistia. Significant depletion at the genus level included Akkermansia and Cloacibacillus. We report increased plasma levels of calprotectin and a higher frequency of gastrointestinal symptoms in PE patients. Furthermore, we report a first description of the gut mycobiome in pregnancy, indicating correlations with gestational length and BMI. Our results suggest that gut dysbiosis might play a role in the pathophysiology of PE and that gastrointestinal symptoms might be a previously overlooked manifestation of the maternal syndrome and a potential target for therapy.
We are studying an association between a disease and microbial profiles, but everything is complicated by the simultaneous state of pregnancy, for which a change in the intestinal bacterial microbiome has already been described.
Physiologically, gut the microbiota of pregnant women is dominated by two major bacterial phyla, Firmicutes and Bacteroidetes, followed by Actinobacteria, Proteobacteria, and Verrucomicrobia. This compositional pattern of bacterial phyla is generally seen in the human gut microbiota and is widely observed in normal non-pregnant adult populations, despite the occasional perturbance of low-abundance phyla. Healthy pregnant women exhibit a consistent enterotype that is clearly driven by the abundance of several dominant genera, such as Bacteroides, Prevotella, and Ruminococcus. This enterotype composition has various fluctuations across all gestational stages, with a slight reduction in the Ruminococcus enterotype in the last stage of pregnancy. Four genera, Ruminococcus, Collinsella, Megamonas, and unclassified Erysipelotrichaceae, increase continuously with gestational age, whereas Ruminococcus, Dialister, and unclassified Lachnospiraceae decrease continuously [bib_ref] Systematic analysis of gut microbiota in pregnant women and its correlations with..., Yang [/bib_ref].
Similar to earlier studies on other populations, we observed significant differences in the gut flora composition in PE patients from the south of Sweden. In line with other studies [bib_ref] Early-Onset Preeclampsia Is Associated With Gut Microbial Alterations in Antepartum and Postpartum..., Lv [/bib_ref] [bib_ref] Gut Microbiota Dysbiosis and Increased Plasma LPS and TMAO Levels in Patients..., Wang [/bib_ref] , we observed a significant increase in Bacteroidetes in PE. Wang et al. also showed increased blood and feces levels of LPS in PE [bib_ref] Gut Microbiota Dysbiosis and Increased Plasma LPS and TMAO Levels in Patients..., Wang [/bib_ref]. As a major contributor to LPS biosynthesis, increased Bacteroidetes abundance can increase inflammation through increased expression of TNF and IL-6, which are known to be elevated in PE through TLR4 [bib_ref] Inflammatory Markers and Preeclampsia: A Systematic Review, Black [/bib_ref]. Injections of LPS in pregnant rodents have been shown to cause PE-like symptoms [bib_ref] Inflammation in rat pregnancy inhibits spiral artery remodeling leading to fetal growth..., Cotechini [/bib_ref] [bib_ref] Activation of the cholinergic anti-inflammatory pathway by nicotine ameliorates lipopolysaccharide-induced preeclampsia-like symptoms..., Liu [/bib_ref].
The genus Akkermansia, belonging to the phylum Verrucomicrobia, was significantly depleted in PE, in line with other studies [bib_ref] Early-Onset Preeclampsia Is Associated With Gut Microbial Alterations in Antepartum and Postpartum..., Lv [/bib_ref] [bib_ref] Gut dysbiosis induces the development of pre-eclampsia through bacterial translocation, Chen [/bib_ref]. Akkermansia is almost the only genus in the phylum Verrucomicrobia, both of which appeared to be significantly reduced in the LEfSe analysis. This may define it as the most representative genus of the Verrucomicrobia phylum depletion in PE. The significant changes at different taxonomic levels in the LEfSe analysis likely also included similar overlaps in some of the other taxa. Akkermansia is involved in gut permeability homeostasis, based on its mucin-degrading ability [bib_ref] Intestinal Integrity and Akkermansia muciniphila, a Mucin-Degrading Member of the Intestinal Microbiota..., Collado [/bib_ref] [bib_ref] Akkermansia muciniphila gen. nov., sp. nov., a human intestinal mucin-degrading bacterium, Derrien [/bib_ref] , and Akkermansia muciniphila is associated with a low risk of diabetes [bib_ref] An increase in the Akkermansia spp. population induced by metformin treatment improves..., Shin [/bib_ref] , obesity [bib_ref] Cross-talk between Akkermansia muciniphila and intestinal epithelium controls diet-induced obesity, Everard [/bib_ref] , and high levels of inflammation [bib_ref] Commensal Akkermansia muciniphila Exacerbates Gut Inflammation in Salmonella Typhimurium-Infected Gnotobiotic Mice, Ganesh [/bib_ref]. At least one study indicated increased gut permeability in PE using the gut permeability marker zonulin [bib_ref] Zonulin as marker of pregnancy induced hypertension: A case control study, Bawah [/bib_ref]. Moreover, supplementation with A. muciniphila is protective against several cardiometabolic features [bib_ref] Supplementation with Akkermansia muciniphila in overweight and obese human volunteers: A proof-of-concept..., Depommier [/bib_ref]. It is possible that the depletion of Akkermansia in PE patients is coupled with the pathophysiology through increased gut permeability and inflammation.
The genus Cloacibacillus, belonging to the phylum Synergistia, was also significantly depleted in PE. As it is mucin-degrading and SCFA-producing [bib_ref] Cloacibacillus evryensis gen. nov., sp. nov., a novel asaccharolytic, mesophilic, amino-acid-degrading bacterium..., Ganesan [/bib_ref] [bib_ref] Cloacibacillus porcorum sp. nov., a mucin-degrading bacterium from the swine intestinal tract..., Looft [/bib_ref] , Cloacibacillus depletion might have negative effects on intestinal permeability and immune regulation.
The increased frequency of loose/watery stool observed in PE could be associated with the decrease in A. muciniphila and increase in Bacteroidetes. Earlier studies have shown that stool consistency is negatively correlated with species richness and positively correlated with the Bacteroidetes/Firmicutes ratio and that A. muciniphila abundance is positively correlated with colon transit time [bib_ref] Stool consistency is strongly associated with gut microbiota richness and composition, enterotypes..., Vandeputte [/bib_ref].
A tendency towards depletion was seen in the class Actinobacteria in PE, which was present in only one PE patient but all controls. Although we did not perform strain analyses, it is reasonable to believe that the probiotic strain Bifidobacteria made up part of the Actinobacteria depletion, being the largest strain in this class. Bifidobacteria are known for their anti-inflammatory properties, which result from Treg stimulation, increased IL-10 secretion, and the regulation of T helper cell mediated inflammatory responses [bib_ref] Bifidobacteria adolescentis regulated immune responses and gut microbial composition to alleviate DNFB-induced..., Fang [/bib_ref] [bib_ref] Bifidobacterium infantis 35624 administration induces Foxp3 T regulatory cells in human peripheral..., Konieczna [/bib_ref] [bib_ref] Systemic Increase in the Ratio between Foxp3+ and IL-17-Producing CD4+ T Cells..., Santner-Nanan [/bib_ref]. Depletion of the family Bifidobacteriaceae and the genus Bifidobacteria in PE was indicated in a newly published study [bib_ref] Decrease in abundance of bacteria of the genus Bifidobacterium in gut microbiota..., Miao [/bib_ref]. A skewed immune homeostasis because of Bifidobacteria depletion might play a role in the systemic inflammatory response seen in PE.
To the best of our knowledge, this study is the first to provide a description of the gut mycobiome in PE. According to our results, gestational age explains 32.62% of the variation in gut mycobiome composition in the entire cohort. The gut mycobiome is known to play immunomodulatory roles both locally and systemically [bib_ref] Gut Mycobiota in Immunity and Inflammatory Disease, Li [/bib_ref]. The observed differences in mycobiome diversity correlating with BMI and gestational length suggest that the fungal flora, like the bacterial flora, exerts immunomodulatory effects during normal pregnancy. Zygomycota, which were observed in minimal traces in only one PE and one C patient, are known to be under-represented in obesity. Family biodiversity has also been shown to be lower in obesity. Metabolic changes in normal pregnancy are, in some areas, similar to those of obesity, including reduced insulin sensitivity in late pregnancy [bib_ref] Cellular Mechanisms for Insulin Resistance in Normal Pregnancy and Gestational Diabetes, Barbour [/bib_ref].
Our results did not allow us to show any difference in the overall mycobiome diversity in PE compared to controls due to a high rate of retained reads after all amplicon sequence variant detection steps of the pipeline. For this reason, we limited ourselves to charting the fungal profile of all samples of pregnant women. Although there was a significant depletion of the Peniophora genus in PE, it is impossible to ascertain if there was a true difference since so many ASVs were unmeasurable or absent in the samples. The amount of unmeasurable or absent ASVs was related to known difficulties in ITS sequencing, e.g., the fact that the gut mycobiome constitutes only 0.1% of the total microbiome [bib_ref] A human gut microbial gene catalogue established by metagenomic sequencing, Qin [/bib_ref]. Moreover, Nilsson and colleagues estimated that more than 10% of the fully identified fungal ITS sequences are incorrectly annotated at the species level [bib_ref] Taxonomic reliability of DNA sequences in public sequence databases: A fungal perspective, Nilsson [/bib_ref]. Fungal communities still represent a poorly studied "black box" for the human gut microbiome, and therefore many individuals do not have a valid classification. Furthermore, it is known that a large number of commensal gut fungi are an environmental signature, rather than commensals whose alteration reflects a difference in the human physiology in health and disease. It is likely that the fungal microbiome is more influenced by environmental factors than by human-physiology-related factors.
In line with other studies [bib_ref] Association of pro-and anti-inflammatory cytokines in preeclampsia, Aggarwal [/bib_ref] [bib_ref] Calprotectin in pregnancy and pregnancy-associated diseases: A systematic review and prospective cohort..., Rezniczek [/bib_ref] , we report a significant increase in calprotectin plasma levels in PE, which was expected, considering the systemic inflammatory response in PE. However, we could not see any differences in the fecal levels of calprotectin, indicating that there might not be any significant local inflammatory response in the gas-trointestinal tract per se. Some individuals (three controls and four PE patients) displayed high values, and a high level of variation was seen between subjects, especially in the PE group. The majority of patients displaying high values had early-onset PE. It is possible that subgroups within PE display gastrointestinal involvement as a feature. However, high feces calprotectin did not correlate with more frequent bowel movements or loose/watery stool.
Although the blood glucose levels obtained in the standard OGTT showed significantly lower values in patients who later developed PE, it is unclear whether this has any clinical relevance in general or for the gut microbiota specifically. All levels were within normal reference values. Diabetes mellitus is associated with gut dysbiosis [bib_ref] Metabolic endotoxemia initiates obesity and insulin resistance, Cani [/bib_ref] [bib_ref] Involvement of gut microbiota in the development of low-grade inflammation and type..., Cani [/bib_ref] [bib_ref] Linking Gut Microbiota and Inflammation to Obesity and Insulin Resistance, Saad [/bib_ref]. High blood glucose, or gestational diabetes mellitus, is a risk factor for PE [bib_ref] The hypertensive disorders of pregnancy: ISSHP classification, diagnosis & management recommendations for..., Brown [/bib_ref]. However, there are no studies indicating that lower levels would also be a risk factor. The patients with the earliest onset of PE manifested before they underwent the OGTT, and therefore we do not have values from these patients.
The role played by the gut microbiome in PE and pregnancy in general is still poorly understood, and further studies are needed. This study provides a glimpse into the gut mycobiome in pregnancy, indicating that there might be composition changes during pregnancy. There is also a need for a more in-depth analysis with targeted metagenomics to determine the strain-specific changes in taxa that are differentially abundant in PE.
In summary, we suggest that increased Bacteroidetes and depleted Synergistia, Akkermansia, and possibly Actinobacteria might play a role in PE pathophysiology. This might be mediated through the combined effects of decreased gut membrane permeability, increased LPS signaling, decreased SCFA production, decreased Treg activity, and an increased T1 inflammatory response. The gut mycobiome might also be an important player in immunomodulatory changes in pregnancy. However, in this cohort the mycobiome was not altered in PE. Interestingly, the data also suggest that increased bowel movements and loose/watery stool might be a part of the maternal syndrome of PE, something that clinicians should pay more attention to in the clinical management of PE.
Supplementary Materials: The following supporting information can be downloaded at https: //www.mdpi.com/article/10.3390/biom13020346/s1, : Summary of questionnaire answers regarding gastrointestinal symptoms; [fig_ref] Figure 1: Plasma [/fig_ref] : Plasma and feces calprotectin levels after excluding outliers. Institutional Review Board Statement: This study was conducted in accordance with the Declaration of Helsinki and was approved by the Regional Ethics Committee of Lund University (2017/419).
# Informed consent statement:
Informed consent was obtained from all subjects involved in the study.
Data Availability Statement: Raw sequencing data were deposited in the European Nucleotide Archive (ENA) under the accession code PRJEB59212.
## Conflicts of interest:
The authors declare no conflict of interest.
[fig] Figure 1: Plasma (a) and feces (b) calprotectin levels in controls vs. PE. (a) PE: N = 25, median 3764 µg/L, IQR 2897-13,639 µg/L; C: N = 28, median 1819 µg/L, IQR 1176-2612 µg/L. (b) PE: N = 20, median 21.0 mg/kg, IQR 8.4-66.2 mg/kg; C: N = 26, median 29.1 mg/kg, IQR 21.2-56.9 mg/kg. The line indicates the upper normal reference level for fecal calprotectin. Mann-Whitney U-test for significance, *** p < 0.001. [/fig]
[fig] Figure 2: Diversity indices for fungal and bacterial microbiome composition in control and PE pregnancies, based on ITS and 16S sequencing. Beta diversity illustrated with principal coordinate analysis (PCoA) for fungal (a) and bacterial microbiota (b). Vectors indicate increasing BMI and gestational age. (c) The two bar plots quantitatively depict how BMI and gestational age influence the differences between the two groups BMI+ and BMI-, separated in the PCoA plot for fungal microbiota (a). C: control, PE: preeclampsia, l.o: lacto-ovo vegetarian, omn: omnivorous, veg: vegan diet. T-test for significance. * p = 0.0361. However, six samples, including both controls and PE, were clustered distant from the other samples in the upper left quadrant in the fungal PCoA plot [/fig]
[fig] Figure 3: Stacked bar plots of the relative abundance with the standard deviation of bacteria in the gut microbiota in 24 control and 15 PE pregnancies, based on successfully sequenced s ples of the 16S rRNA gene. Abundances at the phylum level in individual samples (a). Mean dances according to group at the class level, with colors indicating phyla (b). Cutoff for taxo clusion = 0.005%. [/fig]
[fig] Figure 4: Linear discriminant analysis effect size (LEfSe) analysis showing significantly differential relative abundances of bacteria in control and PE pregnancies at all taxonomic levels. LDA score threshold = 3. Green = higher in PE, red = higher in controls. Results with p values < 0.05 were considered significant and are shown in the figure. [/fig]
[fig] Figure 5: Bar and stacked bar plots showing relative percentage abundances of mycobiome taxonomic bins in ITS gene sequence datasets: the phylum (a) and class levels (b) are shown individually, while the species level is shown as means across the cohort (c). The most abundant fungal species are displayed. Cutoff for taxon inclusion = 0.01%. [/fig]
[fig] Funding: This project was funded by the Swedish Research Council (grant number 314373.35.135949), ALF, Region Skåne, and the SUS Foundations. [/fig]
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Oscillatory ventilation redux: alternative perspectives on ventilator-induced lung injury in the acute respiratory distress syndrome
For patients with the acute respiratory distress syndrome (ARDS), ventilation strategies that limit end-expiratory derecruitment and end-inspiratory overdistension are the only interventions to have significantly reduced the morbidity and mortality. For this reason, the use of high-frequency oscillatory ventilation (HFOV) was considered to be an ideal protective strategy, given its reliance on very low tidal volumes cycled at very high rates. However, results from clinical trials in adults with ARDS have demonstrated that HFOV does not improve clinical outcomes. Recent experimental and computational studies have shown that oscillation of a mechanically heterogeneous lung with multiple simultaneous frequencies can reduce parenchymal strain, improve gas exchange, and maintain lung recruitment at lower distending pressures compared to traditional 'single-frequency' HFOV. This review will discuss the theoretical rationale for the use of multiple oscillatory frequencies in ARDS, as well as the mechanisms by which it may reduce the risk for ventilator-induced lung injury.
Oscillatory ventilation redux: alternative perspectives on ventilator-induced lung injury in the acute respiratory distress syndrome David W [bib_ref] Functional disability 5 years after acute respiratory distress syndrome, Herridge [/bib_ref] [bib_ref] Definition and epidemiology of acute respiratory distress syndrome, Rezoagli [/bib_ref] For patients with the acute respiratory distress syndrome (ARDS), ventilation strategies that limit end-expiratory derecruitment and end-inspiratory overdistension are the only interventions to have significantly reduced the morbidity and mortality. For this reason, the use of high-frequency oscillatory ventilation (HFOV) was considered to be an ideal protective strategy, given its reliance on very low tidal volumes cycled at very high rates. However, results from clinical trials in adults with ARDS have demonstrated that HFOV does not improve clinical outcomes. Recent experimental and computational studies have shown that oscillation of a mechanically heterogeneous lung with multiple simultaneous frequencies can reduce parenchymal strain, improve gas exchange, and maintain lung recruitment at lower distending pressures compared to traditional 'single-frequency' HFOV. This review will discuss the theoretical rationale for the use of multiple oscillatory frequencies in ARDS, as well as the mechanisms by which it may reduce the risk for ventilator-induced lung injury.
# Introduction
Of the many pathologies associated with respiratory failure, the acute respiratory distress syndrome (ARDS) is perhaps the most devastating in terms of outcome. Respiratory failure from ARDS is associated with mortality approaching 40% [bib_ref] Incidence and outcomes of acute lung injury, Rubenfeld [/bib_ref]. Survivors may also be burdened with substantial morbidity, including long-term physical and mental health impairments [bib_ref] Functional disability 5 years after acute respiratory distress syndrome, Herridge [/bib_ref]. ARDS thus imposes significant burdens on public health resources worldwide, and only minimal improvements in outcomes have occurred over recent decades [bib_ref] Definition and epidemiology of acute respiratory distress syndrome, Rezoagli [/bib_ref]. Risks for developing ARDS include a diverse range of predisposing factors and initiating insults, such as aspiration, trauma, sepsis, pneumonia, inhalation injury, blood product transfusion, or burns. Regardless of etiology, the syndrome results in a progressive deterioration of lung function towards a final common pathway: hypoxemic respiratory failure characterized by alveolar flooding, derecruitment, reduced compliance, increased shunt fraction, and increased dead space. A key pathologic feature of ARDS is the heterogeneous structural derangements to the lung tissues, arising from inflammation, edema, surfactant dysfunction, and fibroproliferation [bib_ref] Acute respiratory distress syndrome, Matthay [/bib_ref].
Endotracheal intubation and supportive conventional mechanical ventilation (CMV) remain the mainstays of treatment for the early management of ARDS. However CMV may exacerbate existing lung injury, due to cyclic, intratidal overdistention (volutrauma) and repeated, asynchronous opening and closing of airspaces with each inflation (atelectrauma). The mechanical stresses associated with these phenomena, as well as the temporal rates at which they are applied [bib_ref] Role of strain rate in the pathogenesis of ventilator-induced lung edema, Protti [/bib_ref] , result in the release of cytokines and other inflammatory mediators (biotrauma) that can further exacerbate the existing injury [bib_ref] On the physiologic and clinical relevance of lung-borne cytokines during ventilatorinduced lung..., Dreyfuss [/bib_ref] [bib_ref] Ventilator-induced lung injury, Slutsky [/bib_ref]. This ventilator-induced lung injury (VILI) is thus a direct result of the mechanical heterogeneity of injured parenchyma, leading to maldistribution of ventilation and corresponding impairments in gas exchange. Since ventilation distribution in ARDS is governed by a heterogeneous distribution of regional mechanics, the most appropriate distending pressure, ventilation frequency, or tidal volume for one lung region may not necessarily be the same for another, even within the same patient. This conundrum of minimizing injurious stretch, while maintaining life-supporting gas exchange, is central to the ventilator management of these complex patients [bib_ref] Intratidal overdistention and derecruitment in the injured lung: s simulation study, Amini [/bib_ref].
Ventilation strategies that limit this end-expiratory derecruitment and end-inspiratory overdistension are the only interventions to have significantly reduced the morbidity and mortality of ARDS, using low tidal volume (V T ) or driving pressure (DP) to reduce inspiratory overdistention [bib_ref] Ventilation with lower tidal volumes as compared with traditional tidal volumes for..., Brower [/bib_ref] [bib_ref] Driving pressure and survival in the acute respiratory distress syndrome, Amato [/bib_ref] , and appropriate levels of positive end-expiratory pressure (PEEP) to limit end-expiratory opening and closing [bib_ref] Atelectrauma versus volutrauma: a tale of two time-constants, Bates [/bib_ref]. Such 'protective' ventilation strategies, however, may result in significant hypoventilation of the injured lung. Increasing respiratory rate is the only means available to increase CO 2 removal during CMV, but may be largely ineffective given the increased dead space and ventilation-to-perfusion mismatch associated with ARDS [bib_ref] Pulmonary dead-space fraction as a risk factor for death in the acute..., Nuckton [/bib_ref]. Adjustments to V T , DP, or PEEP based on such criteria provide little insight into how such interventions impact regional gas transport in the injured lung, or how to customize ventilator management for the pathophysiology of an individual patient. For example, optimal PEEP for a given patient depends much more on the unique pattern of injury and the amount of recruitable lung [bib_ref] Lung recruitment in patients with the acute respiratory distress syndrome, Gattinoni [/bib_ref] , rather than on oxygenation alone [bib_ref] The National Heart, Lung, and Blood Institute ARDS Clinical Trials Network: Higher..., Brower [/bib_ref]. Moreover, there may still exist focal regions of high stress and strain within the lung, despite apparently modest distending pressures transduced at the airway opening [bib_ref] Stress distribution in lungs: a model of pulmonary elasticity, Mead [/bib_ref] [bib_ref] The POOR get POORer: a hypothesis for the pathogenesis of ventilator-induced lung..., Gaver Dp 3rd [/bib_ref]. Thus the ability to improve, if not optimize, non-injurious ventilation in patients with ARDS is a consideration not only for improving in clinical outcomes, but also for appropriate management of scarce resources.
## The rise and fall of high frequency oscillatory ventilation in ards
High-frequency oscillatory ventilation (HFOV) is an alternative form of mechanical ventilation utilizing tidal volumes smaller than anatomic dead space, 10-fold to 50-fold higher respiratory frequencies than CMV, and high instantaneous flows. Compared to CMV which relies on convective transport as the dominant mechanism for gas exchange [17], HFOV relies on several different mechanisms, including turbulence, pendelluft, asymmetric velocity profiles, Taylor dispersion, molecular diffusion, collateral ventilation, and cardiogenic mixing . The relative contributions for any of these gas transport mechanisms are highly dependent on the frequency and amplitude of oscillation, the branchingmorphometryoftheairwaytree,aswellasthesize and mechanical properties of the lung . HFOV thus appears to achieve many of the goals of a lung protective strategy, utilizing appropriate mean airway pressures to sustain recruitment, and small tidal volumes to limit overdistention. Consequently, HFOV was initially thought to be panacea for ARDS and VILI . Despite some initial promising results [bib_ref] High-frequency oscillatory ventilation for acute respiratory distress syndrome in adults: a randomized,..., Derdak [/bib_ref] [bib_ref] High frequency oscillation in patients with acute lung injury and acute respiratory..., Sud [/bib_ref] , subsequent large randomized clinical trials and meta-analyses indicated that HFOV did not reduce mortality in adults with ARDS [bib_ref] High-frequency oscillation in early acute respiratory distress syndrome, Ferguson [/bib_ref] [bib_ref] High-frequency oscillation for acute respiratory distress syndrome, Young [/bib_ref] [bib_ref] High-frequency ventilation does not provide mortality benefit in comparison with conventional lung-protective..., Maitra [/bib_ref]. Moreover, HFOV has not been shown to be superior to CMV in preterm or low birth weight infants [bib_ref] Elective high frequency oscillatory ventilation versus conventional ventilation for acute pulmonary dysfunction..., Cools [/bib_ref]. This failure of HFOV to reduce mortality suggested suboptimal, if not injurious, ventilation of the injured lung, potentially arising from variable (and unpredictable) effects of frequency, amplitude, and mean airway pressure [bib_ref] Simulating ventilation distribution in heterogenous lung injury using a binary tree data..., Colletti [/bib_ref] [bib_ref] Heterogeneity of mean alveolar pressure during high-frequency oscillations, Allen [/bib_ref] [bib_ref] Impact of ventilation frequency and parenchymal stiffness on flow and pressure distribution..., Amini [/bib_ref]. The primary determinant of ventilation distribution in the injured lung is the distribution of regional mechanical properties of the airways and parenchyma, such as local resistance, inertance, and elastance [bib_ref] Simulating ventilation distribution in heterogenous lung injury using a binary tree data..., Colletti [/bib_ref]. Even within the same patient, the most appropriate distending pressure, ventilation frequency, or flow/volume amplitude for one region of the lung may not necessarily be the same for another [bib_ref] Did studies on HFOV fail to improve ARDS survival because they did..., Dreyfuss [/bib_ref]. When the lung exhibits such spatial mechanical heterogeneity, local ventilation distribution becomes highly frequency-dependent , and the most effective frequency for optimal gas exchange in a given region will vary depending on its local mechanical properties [bib_ref] Simulating ventilation distribution in heterogenous lung injury using a binary tree data..., Colletti [/bib_ref] [bib_ref] Impact of ventilation frequency and parenchymal stiffness on flow and pressure distribution..., Amini [/bib_ref]. Such frequency-dependence of regional ventilation may also lead to regional hyperinflation and/or derecruitment of the lung in the setting of mechanically heterogeneous disease [bib_ref] Simulating ventilation distribution in heterogenous lung injury using a binary tree data..., Colletti [/bib_ref] [bib_ref] Impact of ventilation frequency and parenchymal stiffness on flow and pressure distribution..., Amini [/bib_ref] [bib_ref] Lung hyperinflation in isolated dog lungs during high-frequency oscillation, Cha [/bib_ref] [bib_ref] Understanding the pressure cost of ventilation: why does high-frequency ventilation work?, Venegas [/bib_ref]. Consistent with this notion, both computational and experimental studies have Oscillatory Ventilation Redux Kaczka 37 Schematic of the concept of multifrequency oscillatory ventilation (MFOV) using a three compartment lung model. Each lung unit preferentially receives convective flows at different oscillatory frequencies, depending on its compliance (or elastance). One unit prefers lower frequencies (a), while the others prefer intermediate (b) or higher (c) frequencies. When these distinct oscillatory frequencies are simultaneously combined into a spectrally broadband MFOV waveform (d), each compartment can selectively 'filter out' its non-preferential frequencies.
demonstrated that oscillatory ventilation at a single high frequency results in some regions of the lung being underventilated and subjected to the risk of atelectrauma, while other regions are overventilated and at risk for volutrauma . Such data indicate that small amplitude volume oscillation at a single, arbitrary frequency is not suitable for maintaining effective gas transport and exchange throughout a spatially heterogeneous lung. In addition, the use of very high mean airway pressures during HFOV may impair venous return and cardiac output, with implications for end-organ perfusion [bib_ref] High-frequency oscillatory ventilation on shaky ground, Malhotra [/bib_ref].
Oscillatory ventilation revisited: the use of multiple simultaneous frequencies Strategies to optimize CMV or HFOV based on arbitrary targets for V T , DP, PEEP, or mean airway pressure neglect the important influence of regional mechanical heterogeneity on ventilation distribution [bib_ref] Simulating ventilation distribution in heterogenous lung injury using a binary tree data..., Colletti [/bib_ref] [bib_ref] Heterogeneity of mean alveolar pressure during high-frequency oscillations, Allen [/bib_ref] [bib_ref] Impact of ventilation frequency and parenchymal stiffness on flow and pressure distribution..., Amini [/bib_ref] [bib_ref] Regional alveolar pressure during periodic flow. Dual manifestations of gas inertia, Allen [/bib_ref]. Recent studies have proposed that mechanical function and gas exchange in the lung can be significantly improved if volume oscillations are applied at multiple frequencies simultaneously, rather than at a single high frequency, due to more even distribution of ventilation to different regions in accordance with local mechanical properties [bib_ref] Multifrequency oscillatory ventilation in the premature lung: effects on gas exchange, mechanics,..., Kaczka [/bib_ref] [bib_ref] Parenchymal strain heterogeneity during oscillatory ventilation: why two frequencies are better than..., Herrmann [/bib_ref] [bib_ref] Strain, strain rate, and mechanical power: an optimization comparison for oscillatory ventilation, Herrmann [/bib_ref] [bib_ref] Quantifying regional lung deformation using four-dimensional computed tomography: a comparison of conventional..., Herrmann [/bib_ref] [bib_ref] Better distribution of minute ventilation and lung energy load in multi-frequency ventilation..., Ort [/bib_ref]. This unique modality has been termed Multi-Frequency Oscillatory Ventilation (MFOV), as a natural extension of HFOV. MFOV is specifically designed to complement the heterogeneity of the injured lung, by relying on the local mechanical impedances of the airways and parenchyma, which can selectively filter out flows of 'less-desirable' frequencies, while simultaneously allowing flows at frequencies more 'optimal' for a particular region to participate in gas exchange. With further adjustments in oscillatory volume amplitude and mean airway pressure, MFOV may improve gas exchange in the injured lung while minimizing the detrimental effects of cyclic alveolar overdistention and derecruitment [bib_ref] Multifrequency oscillatory ventilation in the premature lung: effects on gas exchange, mechanics,..., Kaczka [/bib_ref]. MFOV attempts to exploit mechanical heterogeneity in the lung, by the design and implementation of flow waveforms with spectral content more appropriate for ARDS. Accordingly, MFOV challenges current paradigms for ventilator management in ARDS that seek to reduce the influence of regional mechanical heterogeneity on gas exchange and exacerbation of injury [bib_ref] Effects of inspiratory flow on lung stress, pendelluft, and ventilation heterogeneity in..., Santini [/bib_ref]. [bib_ref] Alveolar pressure nonhomogeneity during small-amplitude high-frequency oscillation, Fredberg [/bib_ref] Physiology of the diseased lung However, its use requires a sophisticated, higher order understanding of the interplay of regional mechanics, ventilation distribution, and gas exchange. Such understanding has been made possible by recent experimental and computer simulation studies.
A previous study in preterm lambs demonstrated that a generic MFOV waveform, generated by a commercially available hybrid pediatric ventilator-oscillator, could achieve significantly better oxygenationand more efficient CO 2 eliminationcompared to traditional single-frequency HFOV in preterm lambs [bib_ref] Multifrequency oscillatory ventilation in the premature lung: effects on gas exchange, mechanics,..., Kaczka [/bib_ref]. These improved indices of gas exchange could also be maintained with significantly lower mean airway pressures, possibly indicating that the additional frequencies in the MFOV waveform enhanced lung recruitment. Moreover, respiratory system elastance was also significantly lower during MFOV compared to HFOV, consistent with enhanced lung recruitment at lower distending airway pressures. These data therefore indicate that MFOV can be a more efficient ventilatory modality in preterm lungs compared to traditional HFOV, and can maintain lung recruitment at lower mean airway pressures. More recently in a dynamic CT imaging study of porcine lung injury [bib_ref] Quantifying regional lung deformation using four-dimensional computed tomography: a comparison of conventional..., Herrmann [/bib_ref] , both HFOV and MFOV improved gas exchange efficiency and reduced intratidal variations in regional strain compared to CMV . These results also indicated that parenchymal strain during oscillatory ventilation was regionally heterogeneous and dependent on frequency. MFOV also significantly reduced the average regional intratidal strain throughout the injured lung compared to either CMV or HFOV, and reduced the spatial gradients of strain compared to CMV.
Consistent with the premise for MFOV, computer simulations in three-dimensional canine, porcine, and human lungs have demonstrated that ventilation distribution and CO 2 elimination is spatially clustered , and highly dependent on both the degree of heterogeneity as well as oscillatory frequency [fig_ref] Figure 4: of a porcine lung with heterogeneous injury [/fig_ref]. Such regional and frequency-dependent differences in gas exchange support the notion that MFOV is ideally suited for the heterogeneously injured lung. More importantly, reduced heterogeneity in the distributions of ventilation and Oscillatory Ventilation Redux Kaczka 39 parenchymal strain can be achieved by the superposition of multiple simultaneous oscillatory frequencies, potentially reducing the risk for VILI compared to traditional single-frequency HFOV [bib_ref] Parenchymal strain heterogeneity during oscillatory ventilation: why two frequencies are better than..., Herrmann [/bib_ref]. It is also theoretically possible that lung-protective MFOV waveforms can be specifically 'tuned' to mechanically heterogeneous lungs of individual patients [bib_ref] Strain, strain rate, and mechanical power: an optimization comparison for oscillatory ventilation, Herrmann [/bib_ref] , with sufficiently low flow/volume amplitudes to reduce parenchymal overdistention, while still being inflated with appropriate distending pressures to reduce cyclic recruitment/derecruitment. However, this likely would require more advanced imaging techniques [bib_ref] Quantifying regional lung deformation using four-dimensional computed tomography: a comparison of conventional..., Herrmann [/bib_ref].
# Conclusions
In the light of recent clinical trials, debates on the merits of oscillatory ventilation as management strategy for ARDS has been tempered [bib_ref] High-frequency oscillation in acute respiratory distress syndrome. The end of the story?, Vincent [/bib_ref] [bib_ref] Meta-analysis of high-frequency oscillation in acute respiratory distress syndrome and accuracy of..., Mentzelopoulos [/bib_ref] , even though it may still hold promise for patients with severe hypoxemia [bib_ref] Severity of hypoxemia and effect of high-frequency oscillatory ventilation in acute respiratory..., Meade [/bib_ref]. Perhaps this indicates a premature abandonment of this very unique, albeit nonintuitive, ventilatory modality in [bib_ref] Understanding the pressure cost of ventilation: why does high-frequency ventilation work?, Venegas [/bib_ref] Physiology of the diseased lung adults [bib_ref] An Official American Thoracic Society/European Society of Intensive Care Medicine/Society of Critical..., Fan [/bib_ref] , before a sufficient understanding of its physiologic risks and benefits could be achieved. Nonetheless, there remains considerable room for improvement in the delivery of oscillatory ventilation in patients [bib_ref] Use of very low tidal volumes during high-frequency ventilation reduces ventilator lung..., Gonzalez-Pacheco [/bib_ref] [bib_ref] Higher frequency ventilation attenuates lung injury during high-frequency oscillatory ventilation in sheep..., Liu [/bib_ref]. By taking advantage of the unique relationship between oscillatory frequency and ventilation distribution in the mechanically heterogeneous lung, MFOV may hold promise as a protective oscillatory ventilation strategy in ARDS and other forms of acute respiratory failure. However, its eventual use in patients will require further preclinical studies to understand its potential applications in other pathophysiologies relevant to ARDS. For example, exacerbations of asthma, COPD, or pneumonia also manifest themselves in a very spatially heterogeneous manner throughout the lung. Thus MFOV may also have implications for the management of acute respiratory failure from other etiologies. The possibility that MFOV can more efficiently penetrate 'difficult-to-reach' regions of the lung also has implications for the optimal delivery of aerosols and drugs, such as beta agonists, steroids, or even inhaled volatile anesthetics [bib_ref] Volatile anesthetics and the treatment of severe bronchospasm: a concept of targeted..., Mondoñ Edo [/bib_ref] [bib_ref] Modeling the dynamics of airway constriction: effects of agonist transport and binding, Amin [/bib_ref]. Moreover, the ability to enhance gas exchange at lower mean airway pressures may make MFOV a more appropriate ventilator modality in patients with impaired cardiac function [bib_ref] The harm of high-frequency oscillatory ventilation (HFOV) in ARDS is not related..., Angriman [/bib_ref] , or in pathologies for which a structurally weakened parenchyma increases the risk barotrauma [bib_ref] High incidence of barotrauma in patients with COVID-19 infection on invasive mechanical..., Mcguinness [/bib_ref] [bib_ref] The ARDSnet protocol may be detrimental in COVID-19, Tsolaki [/bib_ref]. Whether other therapeutic interventions that are known to be efficacious in ARDS, such as prone positioning [bib_ref] Prone positioning in severe acute respiratory distress syndrome, Guerin [/bib_ref] , confer additional lung protection when used in combination with MFOV is of course only speculative at this point. Thus while MFOV may have potential to change current ventilator management in critically ill patients, there remain fundamental questions regarding the mechanisms by which it improves gas exchange and mechanical function. It is also unclear if there are limits on the degree of lung mechanical heterogeneity for which MFOV can still maintain protective ventilation and efficacious gas exchange [bib_ref] Simulating ventilation distribution in heterogenous lung injury using a binary tree data..., Colletti [/bib_ref] [bib_ref] Parenchymal strain heterogeneity during oscillatory ventilation: why two frequencies are better than..., Herrmann [/bib_ref] [bib_ref] Strain, strain rate, and mechanical power: an optimization comparison for oscillatory ventilation, Herrmann [/bib_ref] [bib_ref] Regional gas transport during conventional and oscillatory ventilation assessed by xenon-enhanced computed..., Herrmann [/bib_ref]. The preliminary experimental and computational studies presented in this review suggest that MFOV has potential to reduce the risk of VILI compared to CMV or traditional HFOV, at least based on short term physiologic and mechanical metrics. The potential for MFOV or other oscillatory modalities to reduce the morbidity and mortality associated with ARDS must of course await future clinical trials.
# Funding
This work was supported in part by the Department of Anesthesia at the University of Iowa Hospital and Clinics and National Institutes of Health award R41 HL140640.
## Conflict of interest statement
Dr Kaczka is a co-founder and shareholder of OscillaVent, Inc., and is a co-inventor on a patent involving multifrequency oscillatory ventilation.
## References and recommended reading
[fig] Figure 4: of a porcine lung with heterogeneous injury. The airway tree is shown in black (a), while the colored acini denote tidal strain (b), mechanical power (c), and CO 2 elimination (d) throughout the model during oscillatory ventilation at 10 Hz and 46.7 mL. These oscillatory settings resulted in 5.5 mL min À1 kg À1 of total CO 2 elimination for the whole model. Derecruited acini are shown in gray ($39% of all acini in the model). Direction of gravity g is into the page () or toward the bottom of the page (#) as indicated. Modified from Ref.[46], with permission. [/fig]
[fig] 24: Ip T, Mehta S: The role of high-frequency oscillatory ventilation in the treatment of acute respiratory failure in adults. Curr Opin Crit Care 2012, 18:70-79. 25. Derdak S: High-frequency oscillatory ventilation for acute respiratory distress syndrome in adult patients. Crit Care Med 2003, 31:S317-S323. 26. Derdak S: High-frequency oscillatory ventilation for adult acute respiratory distress syndrome: a decade of progress. Crit Care Med 2005, 33(3 Suppl):S113-S114. [/fig]
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The paradigm of drug resistance in cancer: an epigenetic perspective
chemotherapy favors inherently drug-resistant cells (for instance, cancer stem cells (CSCs)) to self-renew, proliferate, and even undergo epithelial-to-mesenchymal transition (EMT) that promotes their metastatic dissemination [5][6][7]. The tumor cells can also employ intricate signaling cascades to acquire chemoresistant properties during therapy [8,9]. These inherent and acquired chemoresistant cells serve as a repertoire for future tumor recurrence that can give rise to even more aggressive forms of the disease.The concept of drug resistance was first reported in bacteria, based on antibiotic resistance. Studies on cancer and several other diseases showed similar, evolutionarily conserved mechanisms that impart resistance towards treatment stratagems [10]. For example, therapy resistance in cancer was first noted in the 1940s [11], and over the years, it has become one of the significant concerns of present day cancer management. In addition, a thorough understanding of critical molecular and epigenetic pathways behind this drug-resistant property of cancer cells could unveil a magnitude of possibilities that can be employed to sensitize these resistant cells towards conventional treatment regimens successfully. Finally, a comprehensive overview of the current understanding of the epigenetic mechanisms that render therapy resistance in cancer cells is also elucidated through this review.CSCs: the key players behind drug resistanceThe recent era of cancer research has shifted a significant focus towards CSCs as they are being reported as one of the major culprits behind maintaining all the hallmarks of cancer. CSCs, a distinct subset of cells within the heterogeneous tumor mass, are adorned with the ability to self-renew and differentiate into multiple lineages[12]. Over the past few years, many studies have established that CSCs are primary therapy-resistant cells within a tumor, which show numerous resistance mechanisms towards radiotherapy and conventional chemotherapeutic regimen[13,14]. The CSCs are usually resistant to chemotherapy/radiotherapy, which drives as a significant cause of cancer recurrence. They survive after the therapeutic intervention, serving as the seed to promote future tumor relapse. Neoadjuvant chemotherapy enriches the CSC population, suggesting a higher degree of resistance to therapy than the rest of the bulk tumor[15]. Interestingly, CSCs are reported to display different mechanisms of therapy resistance, including induction of EMT, high expression of ATP-binding cassette transporters (ABC transporters) and detoxification genes, quiescence, and evasion of apoptosis[16,17].EMTThe EMT is the preliminary step in cancer metastasis whose induction is governed by epigenetic modifiers, like in the case of one of the transcription factors, Snail, which recruits multiple chromatin modifiers such as PRC2, histone deacetylase (HDAC)1/2, G9a, LSD1, to the promoter of E-cadherin, thereby promoting Snail-mediated E-cadherin repression, a hallmark of EMT[18].It has been reported that the induction of EMT promotes the acquisition of stem cell-like features in cancer cells. For instance, Twist, Slug, and Snail, the critical regulators of EMT, induce mesenchymal properties in breast cancer cells, triggering stem cell-like properties and mammosphere-forming ability[19]. It has been suggested that EMT is associated with the induction of drug resistance [7,20]. Previous studies have indicated that the distinct phenotypic differences between CSCs and non-stem cancer cells (NSCCs) are majorly due to the induction of EMT and mesenchymal properties in the CSCs [6]. However, the conventional therapeutic regimens fail to eliminate the cancer cells with CSC-like and mesenchymal properties, promoting CSC-mediated tumor recurrence [7].Interestingly, EMT and stemness share common signaling pathways like Wnt, Hedgehog, and Notch [20], which suggest their standard mode of action in the induction of drug resistance[21,22]. For example, TGF-β-induced EMT promotes drug resistance [23], while inhibition of TGF-β sensitizes the cells towards chemotherapy[24,25]. Notch 1 signaling-induced EMT activation triggers gefitinib resistance in lung cancer cells[26,27]. The transcription factor ZEB1, which contributes to the regulation of stemness, also plays a significant role in inducing chemoresistance[28]. In pancreatic ductal adenocarcinoma cells, loss of Twist and Snail sensitizes them towards gemcitabine, improving the prognostic outcome[29]. Finally, mechanistically stemness, EMT, and drug resistance are linked, and understanding this network is crucial for developing more potent therapeutic strategies.Increased expression of ABC transporter and detoxification genesABC transporters like ABCB1 (MDR1), ABCC1 (MRP1), ABCG2 are transmembrane proteins that pump toxins out of the cells using energy from ATP hydrolysis. CSCs of various solid tumors display significantly high expression of these drug efflux pumps, rendering them intrinsically resistant to most conventional therapeutic interventions[30,31]. ABCG2 has been reported to efflux the chemotherapeutic drugs doxorubicin and methotrexate [32] and its down-regulation enhances the chemosensitivity of breast CSCs[33]. Importantly, in glioma tumor stem-like cells, Bioscience Reports (2022) 42 BSR20211812 https://doi.org/10.1042/BSR20211812PI3K/Akt signaling promotes localization of ABCG2 to the plasma membrane[34]. Moreover, CSC marker aldehyde dehydrogenase 1 (ALDH1), which catalyzes the oxidation of aldehydes, protects them from reactive oxygen species (ROS)-mediated damages[35]. It is also reported that lung carcinoma stem cells showing elevated expression of ALDH1 confer resistance towards gefitinib, an epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) and other chemotherapeutic drugs[36].Dormancy or quiescenceChemotherapy and radiation therapy effectively damages the proliferating tumor cells. However, they have little effect on the cells in dormant/quiescent state[37]. The quiescent state is a state of reversible cell cycle arrest, where the cells remain in the G 0 phase and require a mitogen stimulus to enter into the division phase[38]. In the core tumor, CSCs often remain in the state of dormancy, which helps them avoid being targeted by drugs and may give them time for efficiently repairing the damaged DNA. Interestingly, in a study in the human bladder cancer xenograft model, between the gap of chemotherapy cycles, quiescent-label retaining CSCs got recruited into cell division in response to the drug-induced damage. This shows similarity to the recruitment of normal stem cells during the wound-healing process and indicates the role of CSCs in the development of therapy resistance[39].
Innate and acquired resistance towards the conventional therapeutic regimen imposes a significant challenge for the successful management of cancer for decades. In patients with advanced carcinomas, acquisition of drug resistance often leads to tumor recurrence and poor prognosis after the first therapeutic cycle. In this context, cancer stem cells (CSCs) are considered as the prime drivers of therapy resistance in cancer due to their 'non-targetable' nature. Drug resistance in cancer is immensely influenced by different properties of CSCs such as epithelial-to-mesenchymal transition (EMT), a profound expression of drug efflux pump genes, detoxification genes, quiescence, and evasion of apoptosis, has been highlighted in this review article. The crucial epigenetic alterations that are intricately associated with regulating different mechanisms of drug resistance, have been discussed thoroughly. Additionally, special attention is drawn towards the epigenetic mechanisms behind the interaction between the cancer cells and their microenvironment which assists in tumor progression and therapy resistance. Finally, we have provided a cumulative overview of the alternative treatment strategies and epigenome-modifying therapies that show the potential of sensitizing the resistant cells towards the conventional treatment strategies. Thus, this review summarizes the epigenetic and molecular background behind therapy resistance, the prime hindrance of present day anti-cancer therapies, and provides an account of the novel complementary epi-drug-based therapeutic strategies to combat drug resistance.
## Drug resistance, a vile opponent to current anti-cancer therapies
In 1971, 'National Cancer Act' of the U.S. Congress declared a 'War on cancer.' Since then, billions of dollars have been invested in cancer research. Yet, despite decade-long studies and advancement in anti-cancer therapeutic regimens, cancer still poses as one of the major causes of global morbidity and mortality, with the diagnosis of millions of new cases every year. In addition, the development of drug resistance, consequent ineffectiveness of therapy, and successive tumor relapse lead to poor predictive outcomes in the majority of the cases and serve as the major limiting factors of the present day anti-cancer therapies. Studies have shown that despite substantial development in the early detection of breast cancer, more than one-third of patients do not respond to the primary chemotherapeutic regimens and develop therapy resistance .
Moreover, the fact that chemotherapy is used as the first-line treatment strategy against the majority of the advanced carcinomas only worsens the scenario . Despite the effective reduction in tumor size at the primary site, several patients display the development of distant metastasis , which is frequently associated with the development of chemoresistance. It is reported that the selection pressure induced by Moreover, the increased expression of ABCG2 and the reduced folate carrier SLC19A1 is reported to be due to promoter hypomethylation, which positively correlates with induction of drug resistance [bib_ref] Chemotherapeutic drug-induced ABCG2 promoter demethylation as a novel mechanism of acquired multidrug..., Bram [/bib_ref] [bib_ref] Aberrant methylation in the promoter region of the reduced folate carrier gene..., Ferreri [/bib_ref]. Furthermore, the expression of Phase-III transporters such as ABCC6 and SLC22A3 is also modulated by alterations in DNA methylations [bib_ref] Epigenetic regulation of drug metabolism and transport, Peng [/bib_ref]. Interestingly, in triple-negative breast cancer, the chromatin reader and tumor suppressor protein ZMYND8 promote the formation of a transcription repressor complex with KDM5C and EZH2 that increases the H3K27me3 mark on promoters of ABCB1, ABCC1, and ABCC2 drug efflux pumps, thereby repressing their expression [bib_ref] Suppression of poised oncogenes by ZMYND8 promotes chemo-sensitization, Mukherjee [/bib_ref]. These pieces of evidence indicate the ability of CSCs to promote drug resistance by epigenetic modifications of DNA and chromatin landscape for up-regulating drug efflux pump expression.
## Epigenetic regulation in drug detoxification
Drug-metabolizing enzymes (DMEs), including phase-I and phase-II DMEs, are responsible for biotransformation and detoxification. In phase-I reaction, the drugs are hydrolyzed and detoxified by DMEs like cytochrome P450 enzymes, aldehyde dehydrogenases etc. [bib_ref] Epigenetic regulation of differentially expressed drug-metabolizing enzymes in cancer, Wang [/bib_ref]. While phase-II DMEs catalyze the conjugation of the hydrophilic compound with the products of phase-I reaction making the latter water-soluble compound for easy excretion. Phase-II DMEs consist of transferases such as glutathione S-transferase (GST) [bib_ref] Epigenetic regulation of differentially expressed drug-metabolizing enzymes in cancer, Wang [/bib_ref]. Epigenetic interplay differentially regulates the DME genes in cancer, mediating their drug resistance potential. In addition, CSCs display a significant increase in drug detoxification machineries, promoting their 'non-targetable' nature. CSCs are associated with high expression of detoxifying enzymes like ALDH1, which is used to identify CSCs [bib_ref] Aldehyde dehydrogenase as a marker for stem cells, Moreb [/bib_ref]. A report by Honoki et al. demonstrated that the CSCs residing in tumorospheres possess high ALDH1 activity and show the robust capability of chemoresistance and drug detoxification [bib_ref] Possible involvement of stem-like populations with elevated ALDH1 in sarcomas for chemotherapeutic..., Honoki [/bib_ref] [fig_ref] Figure 1: The epigenetic regulations behind the different mechanisms of drug resistanceSchematic diagram representing... [/fig_ref]. Another recent study by Peitzsch et al. demonstrated that chemotherapy and irradiation lead to an enrichment of H3K36me3 transcription activation mark on ALDH1 promoter, which up-regulates ALDH1 expression and promotes resistant CSC-like phenotype in the treated cells [bib_ref] An epigenetic reprogramming strategy to resensitize radioresistant prostate cancer cells, Peitzsch [/bib_ref].
P450 is an abundant DME family, subdivided into two subfamilies CYP1-4 and CYP7-51 [bib_ref] Epigenetic regulation of differentially expressed drug-metabolizing enzymes in cancer, Wang [/bib_ref]. Genome-wide integrative analysis established that the DME genes such as CYP2D6, SULT1A1, GSTM5, CYP2C19, CYP1A2, GSTT1, and GSTA4 are differentially modulated by DNA methylation in cancer which leads to inter-and intra-individual differences in drug metabolism, ultimately correlating to poor prognostic outcome. Interestingly, in prostate cancer, CYP24A1, another cytochrome P450 family enzyme, is repressed by promoter methylation and histone repressor mark H3K9me2 [bib_ref] Epigenetic regulation of vitamin D 24-hydroxylase/CYP24A1 in human prostate cancer, Luo [/bib_ref]. The methylation landscape of the promoters of DMEs predicts prognostic outcome and treatment efficacy of hormone-responsive breast cancers. For instance, the promoter methylation status of estradiol and tamoxifen-metabolizing enzyme CYP1B1 from the P450 family has been reported to predict survival and prognosis in tamoxifen-treated and untreated patients [bib_ref] Association of breast cancer DNA methylation profiles with hormone receptor status and..., Widschwendter [/bib_ref]. Another example in this context is the hypermethylation of a phase-II DME, N-acetyl transferase 1 (NAT1) gene, which regulates tamoxifen resistance in breast cancer [bib_ref] Methylation patterns of genes coding for drug-metabolizing enzymes in tamoxifen-resistant breast cancer..., Kim [/bib_ref].
## The epigenetic mechanisms behind heightened dna repair
The DNA repair pathway responds to DNA damage and maintains genomic integrity. The DNA repair machinery comprises an intricate system of sensors, transducers, and effectors that synchronize the repair of damaged DNA, thus ensuring cellular survival [bib_ref] DNA damage response signaling pathways and targets for radiotherapy sensitization in cancer, Huang [/bib_ref]. The compaction of chromatin regulates the accessibility of DNA repair and other cellular machineries [bib_ref] Chromatin dynamics in genome stability: roles in suppressing endogenous DNA damage and..., Nair [/bib_ref] [bib_ref] Regulation of DNA repair mechanisms: how the chromatin environment regulates the DNA..., Stadler [/bib_ref]. So, by modulating the chromatin compaction, cancer cells modulate the impact of damaging agents over the exposed DNA and subsequently regulate the DNA repair mechanism [bib_ref] Heterochromatin and the DNA damage response: the need to relax, Cann [/bib_ref]. It is indicated that the level of radiation-induced double-strand break (DSB) was 5-50 folds higher in the euchromatin region as compared with the heterochromatin region, and also the euchromatin is more prone towards the regional mutation rate variation [bib_ref] Chromatin compaction protects genomic DNA from radiation damage, Takata [/bib_ref] [bib_ref] Chromatin organization is a major influence on regional mutation rates in human..., Schuster-Böckler [/bib_ref]. Accumulating pieces of evidence of the past few years have indicated cancer cells display altered DNA repair pathways that play a crucial role in their resistance towards genotoxic drugs [bib_ref] The effects of deregulated DNA damage signalling on cancer chemotherapy response and..., Bouwman [/bib_ref]. A study by Kandoth et al. using next-generation sequencing-based large-scale mutation mapping in cancer has unravelled that cancer cells show an elevated rate of gene mutations that play a role in DNA and histone alterations and chromatin remodeling [bib_ref] Mutational landscape and significance across 12 major cancer types, Kandoth [/bib_ref] , thus leading to possible epigenomic alteration in cancer cells. This altered epigenetic landscape in cancer cells leads to global changes in histone modification patterns, DNA methylation, and nucleosome positions, affecting their chromatin architecture [bib_ref] Mutations in regulators of the epigenome and their connections to global chromatin..., Plass [/bib_ref]. These, in turn, alter the DNA damage response and repair in cancer cells, which can either allow accumulation of heightened DNA damage, causing instability in the genome (a hallmark of cancer), or can promote enhanced DNA repair, rendering resistance to genotoxic drugs.
The DNA repair protein Ataxia Telangiectasia Mutation (ATM) functions to sense DNA damage and simultaneously activate the repair mechanism associated with it, including homologous recombination and non-homologous end-joining methods [bib_ref] ATM signaling facilitates repair of DNA double-strand breaks associated with heterochromatin, Goodarzi [/bib_ref]. In addition, ATM is a critical player in DNA DSB repair induced by chemotherapeutic treatment, ionizing radiation etc. In addition, ATM gene aberration and epigenetic alteration of ATM expression in cancer are routinely associated with chemotherapy resistance and poor prognostic outcome [bib_ref] ATM mutations in cancer: therapeutic implications, Choi [/bib_ref]. Hypermethylation in the promoter of hMLH1, a protein involved in mismatch repair, is commonly seen in colorectal cancer [bib_ref] Incidence and functional consequences of hMLH1 promoter hypermethylation in colorectal carcinoma, Herman [/bib_ref] [fig_ref] Figure 1: The epigenetic regulations behind the different mechanisms of drug resistanceSchematic diagram representing... [/fig_ref]. 5-Aza-2 -deoxycytidine (decitabine)-mediated demethylation of hMLH1 gene promoter leads to sensitization of colorectal cancer cells towards chemotherapeutic drug, 5-fluorouracil [bib_ref] Demethylation by 5-aza-2'-deoxycytidine in colorectal cancer cells targets genomic DNA whilst promoter..., Mossman [/bib_ref].
The CSCs show an even more improved rate of DNA repair, which allows them to be the most drug-resistant cells within the heterogeneous tumor mass [bib_ref] Therapy resistance mediated by cancer stem cells, Steinbichler [/bib_ref]. The high expressions of O(6)-methylguanine-DNMT, NBS1, Chk1, and Chk2 in CSCs in comparison to NSCCs renders them more resistant towards DNA-damaging therapies, and the altered expression of these genes in CSCs can be attributed to their differential epigenetic landscape [bib_ref] The cancer stem-cell signaling network and resistance to therapy, Carnero [/bib_ref] [bib_ref] The roles of cancer stem cells and therapy resistance in colorectal carcinoma, Das [/bib_ref].
## The epigenetic allies in inhibiting apoptosis
Tumor development, progression, and response to therapeutic interventions are tightly regulated by programmed cell death or apoptosis [bib_ref] Apoptosis as anticancer mechanism: function and dysfunction of its modulators and targeted..., Pistritto [/bib_ref]. Evading apoptosis is one of the main hallmarks of cancer, ensuring cancer cell survival upon exposure to cytotoxic and genotoxic stresses [bib_ref] Hallmarks of cancer: the next generation, Hanahan [/bib_ref]. Since the mechanism of action of most of the current treatment regimens like chemotherapy is to activate apoptotic pathways, resistance towards apoptosis shapes therapeutic resistance to a massive extent [bib_ref] Apoptosis: a target for anticancer therapy, Pfeffer [/bib_ref]. The acquisition of apoptosis resistance depends on various factors, including equilibrium of pro-and anti-apoptotic signals and expression of the apoptosis-related genes [bib_ref] Epigenetic deregulation of apoptosis in cancers, Ozyerli-Goknar [/bib_ref]. The different pro-and anti-apoptotic signaling factors are regulated epigenetically at transcription and post-translation levels in the cancer cells. For instance, enhanced expression or activity of anti-apoptotic factors like Bcl-xl, Bcl-2, IAPs etc. leads to the development of apoptosis resistance. Similarly, repression of pro-apoptotic factors like Bax, Bid, Puma, Noxa, Bim etc. promotes resistance towards apoptosis. Moreover, Akt and their transcriptional regulators, NF-κB and STAT, remain highly overexpressed in various advanced cancers, indicating their possible involvement in therapy resistance [bib_ref] Evading apoptosis in cancer, Fernald [/bib_ref].
Different histone variants and their modifications are associated with carcinogenesis and therapy resistance. For example, the histone variant H2AZ is reported to be overexpressed in various solid cancers, contributing to tumor progression [bib_ref] Histone variants: emerging players in cancer biology, Vardabasso [/bib_ref]. Knockdown of H2AZ leads to repression of Bcl-2 and elevation of pro-apoptotic marks Caspase 3, Caspase 9, and Bak, thus stimulating apoptotic cell death [bib_ref] H2A.Z regulates tumorigenesis, metastasis and sensitivity to cisplatin in intrahepatic cholangiocarcinoma, Yang [/bib_ref]. Aberrant histone alterations are often associated with cancer progression and therapy resistance. Previous studies have indicated the involvement of dephosphorylation of histone H1, phosphorylation of H2A, H2B, H3, and H4, and deubiquitylation of H2A in the cascade of apoptosis [bib_ref] Histone modifications and apoptosis: cause or consequence?, Th'ng [/bib_ref]. Tumor cells resist drug toxicity-mediated apoptosis by up-regulating the expressions of various DNA repair genes (like MGMT, BRCA 1/2 etc.) through activation of H3K4Me3 [bib_ref] Epigenetic deregulation of apoptosis in cancers, Ozyerli-Goknar [/bib_ref]. A study in human lung cancer cell line NCI-H460 revealed that repression of p16 INK4a promoter by cumulative action of ZBP-89 and HDAC3 regulates senescence and apoptosis [bib_ref] The transcription factor ZBP-89 suppresses p16 expression through a histone modification mechanism..., Lu [/bib_ref] [fig_ref] Figure 1: The epigenetic regulations behind the different mechanisms of drug resistanceSchematic diagram representing... [/fig_ref]. In breast carcinoma, CDKN1C (the gene encoding tumor suppressor p57 KIP2 ) is repressed by EZH2-mediated histone H3K27 trimethylation (H3K27me3), leading to the acquisition of a more resistant phenotype [bib_ref] CDKN1C (p57) is a direct target of EZH2 and suppressed by multiple..., Yang [/bib_ref] [fig_ref] Figure 1: The epigenetic regulations behind the different mechanisms of drug resistanceSchematic diagram representing... [/fig_ref]. In bladder carcinoma cells, the repression of the p21 WAF1 promoter is mediated by histone deacetylation through the activity of HDACs [bib_ref] Histone deacetylase inhibitor selectively induces p21WAF1 expression and gene-associated histone acetylation, Richon [/bib_ref]. This leads to the development of apoptotic resistance, which can be effectively reversed by targeting the cells with HDAC inhibitors (HDACis) [bib_ref] Histone deacetylase inhibitor selectively induces p21WAF1 expression and gene-associated histone acetylation, Richon [/bib_ref]. In addition, specific histone modifications can lead to the recruitment of protein complexes governing the balance between cell death and survival. For example, phosphorylation of H2A.X-Y142 represses MDC1-induced recruitment of DNA repair proteins to DNA damage site (yH2AX) [bib_ref] Histone H2A.X Tyr142 phosphorylation: a novel sWItCH for apoptosis?, Stucki [/bib_ref]. This, in turn, promotes the recruitment of pro-apoptotic complexes to the site, pushing the cell towards apoptotic fate [bib_ref] Histone H2A.X Tyr142 phosphorylation: a novel sWItCH for apoptosis?, Stucki [/bib_ref] [bib_ref] MDC1 directly binds phosphorylated histone H2AX to regulate cellular responses to DNA..., Stucki [/bib_ref]. During the acquisition of therapy resistance, this pathway alters in the cancer cells, thus resisting cell death even after being treated with DNA damaging agents like chemotherapy.
DNA methylation status also plays a pivotal role in modulating apoptotic cell fate in various cancers. The CpG islands on the promoters of tumor suppressor and pro-apoptotic genes remain hypermethylated by the activity of DNMT in cancer cells [bib_ref] DNA hypomethylation in cancer cells, Ehrlich [/bib_ref] [fig_ref] Figure 1: The epigenetic regulations behind the different mechanisms of drug resistanceSchematic diagram representing... [/fig_ref]. Hypermethylation of these genes inhibits the programmed cell death pathway, rendering the tumor cells a resistant phenotype. For instance, the promoter of pro-apoptotic markers Bcl-2 and Bik reportedly remain hypermethylated in prostate cancer [bib_ref] Survey of differentially methylated promoters in prostate cancer cell lines, Wang [/bib_ref] [bib_ref] In silico analysis and DHPLC screening strategy identifies novel apoptotic gene targets..., Murphy [/bib_ref]. Similarly, in multiple myeloma, the promoters of Bad, Bax, Bak, and Puma display hypermethylation [bib_ref] Microarray analysis of epigenetic silencing of gene expression in the KAS-6/1 multiple..., Pompeia [/bib_ref]. The promoter of Bim reportedly shows hypermethylation and repression in chronic myeloid leukemia [bib_ref] Epigenetic down-regulation of BIM expression is associated with reduced optimal responses to..., José-Eneriz [/bib_ref]. Promoter methylation and repression of Caspase 8 and 10, two significant contributors of the apoptotic pathway, render resistant phenotype in bladder cancer, hepatocellular carcinoma, glioblastoma, small-cell lung cancer, retinoblastoma, and neuroblastoma [bib_ref] Methylation patterns of Rb1 and Casp-8 promoters and their impact on their..., Malekzadeh [/bib_ref] [bib_ref] Epigenetic methylation and expression of caspase 8 and survivin in hepatocellular carcinoma, Cho [/bib_ref] [bib_ref] Impact of the DNA methyltransferases expression on the methylation status of apoptosis-associated..., Hervouet [/bib_ref] [bib_ref] Differential inactivation of caspase-8 in lung cancers, Shivapurkar [/bib_ref] [bib_ref] Deregulation of caspase 8 and 10 expression in pediatric tumors and cell..., Harada [/bib_ref]. In gastric cancer, the expression of Bcl-2l10, BNIP3, and HRK remains inhibited by promoter hypermethylation [bib_ref] BCL2L10 protein regulates apoptosis/proliferation through differential pathways in gastric cancer cells, Xu [/bib_ref] [bib_ref] Methylation of BNIP3 and DAPK indicates lower response to chemotherapy and poor..., Sugita [/bib_ref] [bib_ref] Identification of HRK as a target of epigenetic inactivation in colorectal and..., Obata [/bib_ref]. p53, the master tumor suppressor, plays a pivotal role in initiating the apoptotic cascade. In patients with acute lymphatic leukemia, the p53 gene remains hypermethylated and inhibited, leading to the ineffectiveness of treatment regimens [bib_ref] TP53 is frequently altered by methylation, mutation, and/or deletion in acute lymphoblastic..., Agirre [/bib_ref]. Also, repression of hypermethylated in cancer 1 (HIC1) in cancer cells leads to inactivation of p53, thus inhibiting DNA damage-mediated apoptosis pathway [bib_ref] Tumor suppressor HIC1 directly regulates SIRT1 to modulate p53-dependent DNA-damage responses, Wen [/bib_ref]. MGMT, MLH1, BRCA1, APC, and APAF1 expressions are also reported to be diminished in cancer cells by hypermethylation of their promoters [bib_ref] Cancer epigenomics: DNA methylomes and histone-modification maps, Esteller [/bib_ref] [bib_ref] Methylation silencing of the Apaf-1 gene in acute leukemia, Furukawa [/bib_ref]. Repression of FAS expression by hypermethylation in its promoter is reported to be involved in cutaneous T-cell lymphoma carcinogenesis and apoptosis resistance [bib_ref] Reduction of Fas/CD95 promoter methylation, upregulation of Fas protein, and enhancement of..., Wu [/bib_ref].
Apart from DNA methylation and histone post-translational modifications, miRNAs also play a critical role in the modulation of apoptosis resistance. For instance, p53 induces the expression of the miR-34 family, which reportedly can inhibit the expression of MYCN, CDK4/6, Notch, Cyclin E2, and Bcl-2 [bib_ref] 2015) miR-34 and p53: new insights into a complex functional relationship, Navarro [/bib_ref]. The expression of miR-34 is repressed in various solid and liquid cancers, including breast/colon/gastric/kidney/pancreatic carcinoma, Burkitt's lymphoma, and chronic lymphocytic leukemia indicating the ingenious strategy of cancer cells to resist apoptosis [bib_ref] Restoration of tumor suppressor miR-34 inhibits human p53-mutant gastric cancer tumorspheres, Ji [/bib_ref] [bib_ref] Inactivation of miR-34a by aberrant CpG methylation in multiple types of cancer, Lodygin [/bib_ref]. Similarly, the expressions of miR-15, miR-16, miR-193a-3p, miR-29b, miR-133b, miR-512-5p remain repressed in different types of cancers due to their involvement in promoting apoptosis [bib_ref] Histone deacetylases mediate the silencing of miR-15a, miR-16, and miR-29b in chronic..., Sampath [/bib_ref] [bib_ref] Reference miRNAs for miRNAome analysis of urothelial carcinomas, Ratert [/bib_ref] [bib_ref] Epigenetic silencing of microRNA-193a contributes to leukemogenesis in t(8; 21) acute myeloid..., Li [/bib_ref] [bib_ref] Chromatin remodeling at Alu repeats by epigenetic treatment activates silenced microRNA-512-5p with..., Saito [/bib_ref] [bib_ref] MicroRNA 133B targets pro-survival molecules MCL-1 and BCL2L2 in lung cancer, Crawford [/bib_ref] [bib_ref] MiR-153 as a tumor suppressor in glioblastoma multiforme is downregulated by DNA..., Ghasemi [/bib_ref]. In contrast, miR-221 and miR-222 remains overexpressed in various solid cancers, leading to repression of TIMP3, PTEN, FOXO3A, PUMA, Caspase 3, etc. [bib_ref] ) miR-221&222 regulate TRAIL resistance and enhance tumorigenicity through PTEN and TIMP3..., Garofalo [/bib_ref] [bib_ref] MiR-221 and miR-222 target PUMA to induce cell survival in glioblastoma, Zhang [/bib_ref] [bib_ref] CASC2/miR-24/miR-221 modulates the TRAIL resistance of hepatocellular carcinoma cell through caspase-8/caspase-3, Jin [/bib_ref] [bib_ref] MicroRNA-221 silencing predisposed human bladder cancer cells to undergo apoptosis induced by..., Lu [/bib_ref]. miR-BART5 is reported to be involved in the apoptosis-resistance of gastric cancer cells due to its ability to target PUMA [bib_ref] An Epstein-Barr virus-encoded microRNA targets PUMA to promote host cell survival, Choy [/bib_ref]. miR-135a, which reportedly represses JAK2 and Bcl-xl, displays down-regulation in various cancers like ovarian cancer, AML etc. [bib_ref] MicroRNA-135a-3p is downregulated and serves as a tumour suppressor in ovarian cancer..., Duan [/bib_ref] [bib_ref] Downregulation of miR-135a predicts poor prognosis in acute myeloid leukemia and regulates..., Xu [/bib_ref].
All these reports indicate the intricate involvement of epigenetic machinery in modulating apoptosis in cancer cells, indicating that epigenetic reprogramming plays a pivotal role in rendering apoptosis resistance to the cancer cells.
## The role of tumor microenvironment in supporting tumor progression and mediating drug resistance
The tumor microenvironment (TME) is an intricate and dynamic landscape that consists of proliferating tumor cells, tumor-associated immune cells, endothelial cells, and extracellular matrix. The TME plays a crucial part in tumor progression, invasiveness, metastatic insemination and impacts the clinical outcome of the tumor. The interaction between the cell and extracellular matrix of the TME is critical for cancer progression, and it plays a vital role in the development of therapy resistance. The different components of TME are reported to be regulated and reprogrammed by epigenetic alterations, including DNA and histone modifications.
## Role of epigenetics in reprogramming tumor-associated immune landscape
It is well established that epigenetic machinery plays a critical role in reprogramming and recruiting the intrinsic and adaptive immune cells into the TME, supporting tumor progression. It is reported that HDAC5-mediated inhibition of Socs3 and up-regulation of CCL2 leads to recruitment of tumor-associated macrophages (TAMs) [bib_ref] Tumor microenvironment remodeling enables bypass of oncogenic KRAS dependency in pancreatic cancer, Hou [/bib_ref]. In the TAMs, ERK and JNK facilitated histone deacetylation of CIITA promoter leads to decoy receptor (DcR3)-induced repression of MHC-II expression [bib_ref] Epigenetic control of MHC class II expression in tumor-associated macrophages by decoy..., Chang [/bib_ref]. This impairs antigen presentation in TAMs, resulting in immunosuppression in the TME [bib_ref] Epigenetic control of MHC class II expression in tumor-associated macrophages by decoy..., Chang [/bib_ref]. In addition, the myeloid-derived suppressor cells (MDSCs) are a subset of immune cells that expands during cancer and plays a critical role in tumor development, progression, and therapy resistance. It is reported that, in hepatocellular carcinoma, the interaction between EZH2 and p65-NF-kB and their binding on IL-6 promoter augments its expression and induces MDSC recruitment in the TME, which is correlated with a poor therapeutic response [bib_ref] Hepatoma-intrinsic CCRK inhibition diminishes myeloid-derived suppressor cell immunosuppression and enhances immune-checkpoint blockade..., Zhou [/bib_ref] [fig_ref] Figure 1: The epigenetic regulations behind the different mechanisms of drug resistanceSchematic diagram representing... [/fig_ref].
Furthermore, the CD8+ cytotoxic T cells recognize and kill target antigens through MHC-I, which is epigenetically repressed in the TME as a strategy for immune evasion. Finally, a study has reported that PRC2 suppresses MHC-I through bivalent H3K4me3 and H3K27me3 modifications [bib_ref] An evolutionarily conserved function of polycomb silences the MHC class I antigen..., Burr [/bib_ref] [fig_ref] Figure 1: The epigenetic regulations behind the different mechanisms of drug resistanceSchematic diagram representing... [/fig_ref]. Moreover, the expression of DNMT1 and EZH2 shows a negative correlation with CD8+ T-cell infiltration within the TME and consequently the patient's clinical outcome [bib_ref] Epigenetic silencing of TH1-type chemokines shapes tumour immunity and immunotherapy, Peng [/bib_ref].
MicroRNAs also play a distinct role in shaping the immunosuppressed environment within the TMEs, thereby promoting tumor progression. For instance, the repression of miR-29 in cancer promotes augmentation of B7-H3, resulting in dysfunction of Natural Killer (NK) cells and immune evasion [bib_ref] MicroRNA miR-29 modulates expression of immunoinhibitory molecule B7-H3: potential implications for immune..., Xu [/bib_ref]. Also, the inhibition of perforin and granzyme expression by miR-27a* reduces anti-cancer cytotoxic functions of NK cells and cytotoxic T cells within the TME [bib_ref] Human microRNA-27a* targets Prf1 and GzmB expression to regulate NK-cell cytotoxicity, Kim [/bib_ref].
## The role of tme components in tumor progression
A study by Hanson et al. in prostate cancer revealed differential promoter methylation status of various genes in epithelial and stromal cells in benign and malignant tumors [bib_ref] Gene promoter methylation in prostate tumor-associated stromal cells, Hanson [/bib_ref]. It was shown that a distinct promoter methylation pattern in stromal cells is correlated with tumor development. The TME also plays a critical role in inducing intrinsic resistance to chemotherapy via 'reversed pH gradient,' pumping out protons through proton transporters compared with normal cells, which have low intracellular pH compared with extracellular pH [bib_ref] Molecular connections between cancer cell metabolism and the tumor microenvironment, Justus [/bib_ref]. The TME also adapts to dynamic changes as therapy continues, which leads to a gain in resistance to both chemo and targeted drugs. For instance, the CSC component of the TME plays a critical role in developing therapy resistance. The CSCs survive after radio or chemotherapy and shows resistance even towards one of the most novel and selective therapeutic system, immunotherapy [bib_ref] Cancer stem cells (CSCs) in cancer progression and therapy, Najafi [/bib_ref]. The NSCCs, CSCs, and immune components of the TME, through their secretions, reciprocate feedback to each other to overcome therapy-induced stress and increase survival [bib_ref] Drug resistance driven by cancer stem cells and their niche, Prieto-Vila [/bib_ref].
Intricate epigenetic regulatory pathways within the different components of TME play a crucial role in ensuring tumor progression. For example, in TAMs, activating epigenetic marks like histone phosphorylation by ERK-1/2 at the IL-10 promoter results in elevated production of IL-10, which stimulates immunosuppression [bib_ref] TLR signaling-mediated differential histone modification at IL-10 and IL-12 promoter region leads..., Banerjee [/bib_ref]. Moreover, in TAMs, Tet2 reportedly sustains its immunosuppressive and tumor-promoting activity through DNA methylation [bib_ref] The DNA methylcytosine dioxygenase Tet2 sustains immunosuppressive function of tumor-infiltrating myeloid cells..., Pan [/bib_ref]. In triple-negative breast cancer, LSD1 demethylase is allegedly involved in M2 polarization by activating the expression of IL-1b, IL-12b, IL-8, NOS2, CCR7, Gpr18 etc. [bib_ref] LSD1 activation promotes inducible EMT programs and modulates the tumour microenvironment in..., Boulding [/bib_ref]. Also, exosomal miR-21 and miR-1246 can enhance the production of IL-10 and TGF-β in primary human macrophages, leading to M2 activation [bib_ref] Mutant p53 cancers reprogram macrophages to tumor supporting macrophages via exosomal miR-1246, Cooks [/bib_ref] [fig_ref] Figure 1: The epigenetic regulations behind the different mechanisms of drug resistanceSchematic diagram representing... [/fig_ref]. M2 macrophages are established to have pro-tumor functions and have previously been associated with the acquisition of drug resistance [bib_ref] Interaction of tumor-associated macrophages and cancer chemotherapy, Larionova [/bib_ref]. In chemoresistant acute lymphoblastic leukemia, the interaction of cells within the bone marrow microenvironment is reported to be facilitated by epigenetic modifiers. Inhibition of these interactions using DNMT inhibitor azacitidine and HDACi panobinostat reportedly chemosensitizes these cells [bib_ref] Epigenetic drug combination overcomes osteoblast-induced chemoprotection in pediatric acute lymphoid leukemia, Quagliano [/bib_ref].
The components of core TME undergo hypoxic stress that causes elevated expression of hypoxia-inducible factor 1 α (HIF-1α). In hypoxic TME, reduced hydroxylation of HIF-1α favors its stabilization. Also, post-translational modifications of HIF-1α, including phosphorylation and SUMOylation have been reported to promote its stabilization [bib_ref] SUMO-specific protease 1 is essential for stabilization of HIF1alpha during hypoxia, Cheng [/bib_ref]. Overexpression of HIF-1α orchestrates the transcription of various genes leading to the alteration in metabolism, activation of angiogenesis and metastasis, and the development of chemoresistance [bib_ref] Targeting tumor microenvironment for cancer therapy, Roma-Rodrigues [/bib_ref] [bib_ref] The interplay among miRNAs, major cytokines, and cancer-related inflammation, Chakraborty [/bib_ref]. In addition, several microRNAs are reported to play an intricate part in the hypoxia-mediated acquisition of drug resistance.
For instance, down-regulation of miR-15 and miR-16 due to hypoxia is associated with chemoresistance [bib_ref] ) miR-15b and miR-16 modulate multidrug resistance by targeting BCL2 in human..., Xia [/bib_ref] [bib_ref] Hypoxia-microRNA-16 downregulation induces VEGF expression in anaplastic lymphoma kinase (ALK)-positive anaplastic large-cell..., Dejean [/bib_ref]. HIF-1α transcriptionally activates the expression of miR-20a, an elevated level of which has been found to induce therapy resistance [bib_ref] Hypoxia-induced microRNA-20a expression increases ERK phosphorylation and angiogenic gene expression in endometriotic..., Lin [/bib_ref] [bib_ref] miR-20a targets BNIP2 and contributes chemotherapeutic resistance in colorectal adenocarcinoma SW480 and..., Chai [/bib_ref]. Also, hypoxia-regulated expression of miR-200b reportedly promotes chemoresistance in cholangiocarcinoma [bib_ref] Involvement of human micro-RNA in growth and response to chemotherapy in human..., Meng [/bib_ref].
All these studies cumulatively point towards the intricate involvement of epigenetic machinery in reprogramming and utilizing TME in a pro-tumor manner.
## Alternative treatment strategies to sensitize the drug-resistant cells
For advanced stages of cancer, a combination of chemotherapeutic agents is often favored over single agents to achieve a higher and quicker therapeutic response. Combination chemotherapy regimens are reported to show a better response in the case of tumor regression [bib_ref] Single agent versus combination chemotherapy for metastatic breast cancer, Carrick [/bib_ref]. For instance, FAC (5-fluorouracil+adrenamycin+cyclophosphamide) and FEC (5-fluorouracil+adrenamycin+cyclophosphamide) are routinely used combinations for breast cancer. In metastatic breast carcinomas, Gemcitabine has also been utilized with paclitaxel for clinical trials [bib_ref] Gemcitabine plus Paclitaxel versus Paclitaxel monotherapy in patients with metastatic breast cancer..., Albain [/bib_ref]. CHOP (cyclophosphamide, doxorubicin hydrochloride, vincristine, and prednisone) is one of the most routinely used therapy in diffuse-large B-cell lymphoma [bib_ref] Rituximab plus CHOP (R-CHOP) overcomes bcl-2-associated resistance to chemotherapy in elderly patients..., Mounier [/bib_ref]. However, combining two or more chemotherapeutic agents increases the amount of systemic toxicity and increases the chance of chemotherapy-related complications. To address this issue, several studies have utilized other drugs, small molecules, or pathway inhibitors in complement with chemotherapy to sensitize the drug-resistant cells. In recurrent ovarian cancers, VEGF and PARP inhibitors are combined with chemotherapeutic drugs [bib_ref] Therapeutic strategies in epithelial ovarian cancer, Kim [/bib_ref]. The anti-diabetic drugs thiazolidinedione and metformin have been repurposed to target the chemoresistant CSCs by several studies, in which these drugs significantly sensitized the CSCs towards chemotherapy [bib_ref] Thiazolidinediones as anti-cancer agents, Blanquicett [/bib_ref] [bib_ref] Counteracting chemoresistance with metformin in breast cancers: targeting cancer stem cells, Samuel [/bib_ref]. Curcumin, an active component obtained from turmeric (Curcuma longa) rhizome, has been well established by several studies as a potent chemosensitizing agent for various cancers [bib_ref] Curcumin reduces mitomycin C resistance in breast cancer stem cells by regulating..., Zhou [/bib_ref] [bib_ref] Targeting colorectal cancer stem cells using curcumin and curcumin analogues: insights into..., Ramasamy [/bib_ref] [bib_ref] Curcumin inhibits the growth of liver cancer stem cells through the phosphatidylinositol..., Wang [/bib_ref]. The role of NFκB in the acquisition of drug resistance is well recognized by several studies [bib_ref] NF-kappaB transcription factor induces drug resistance through MDR1 expression in cancer cells, Bentires-Alj [/bib_ref] [bib_ref] Nuclear factor-kappa B as a resistance factor to platinum-based antineoplasic drugs, Lagunas [/bib_ref]. The commonly used NSAID aspirin, due to its ability to inhibit nuclear transport of NFκB, has been extensively utilized by several studies to target cancer cells and CSCs [bib_ref] SMAR1 repression by pluripotency factors and consequent chemoresistance in breast cancer stem-like..., Bhattacharya [/bib_ref] [bib_ref] Repositioning aspirin to treat lung and breast cancers and overcome acquired resistance..., Li [/bib_ref] [bib_ref] Aspirin suppresses chemoresistance and enhances antitumor activity of 5-Fu in 5-Fu-resistant colorectal..., Fu [/bib_ref]. Aspirin pre-treatment has been shown to suppress the acquisition of chemoresistance in breast CSCs [bib_ref] Aspirin suppresses the acquisition of chemoresistance in breast cancer by disrupting an..., Saha [/bib_ref]. Quite a handful of studies have pointed to the efficacy of using various miR-NAs combined with chemotherapy to sensitize chemoresistance in cancer [bib_ref] MiRNA-621 sensitizes breast cancer to chemotherapy by suppressing FBXO11 and enhancing p53..., Xue [/bib_ref] [bib_ref] Systematic screen identifies miRNAs that target RAD51 and RAD51D to enhance chemosensitivity, Huang [/bib_ref] [bib_ref] miR-181b modulates multidrug resistance by targeting BCL2 in human cancer cell lines, Zhu [/bib_ref] [bib_ref] Cisplatin sensitivity mediated by WEE1 and CHK1 is mediated by miR-155 and..., Pouliot [/bib_ref]. However, cancer cells' extensive epigenetic reprogramming ability poses a major challenge to all the combinatorial approaches and even increases the possibility of acquiring resistance towards these therapies.
## Combating drug resistance by targeting epigenetic modifiers
Various studies so far have established that in addition to genetic mutations, epigenetic modifications play a pivotal role in acquiring chemoresistance in cancer cells. However, the epigenetic alternations are reversible and require to be actively maintained by epigenetic modifiers, making them an attractive target for therapeutic intervention. The current understanding of CSC epigenome provides new insights into targeted therapy by epigenetic drugs to overcome CSC drug resistance [bib_ref] Current understanding of epigenetics mechanism as a novel target in reducing cancer..., Keyvani-Ghamsari [/bib_ref]. Several studies have utilized epigenome modifying drugs alone or combined with conventional treatments to modulate the epigenetic changes to attenuate drug resistance [bib_ref] Epigenetic therapeutics: a new weapon in the war against cancer, Ahuja [/bib_ref] [fig_ref] Figure 2: Epidrugs are used to target and sensitize CSCs Several studies have utilized... [/fig_ref]. Although these epigenome-altering drugs are 'non-specific' in nature and may affect global gene expression, it is also reported that they can cause local changes in gene expression depending on the gene chromatin environment [bib_ref] Loci specific epigenetic drug sensitivity, Zhang [/bib_ref]. Furthermore, it is reported that the sensitivity towards epigenetic alternators can be genomic loci-specific depending on the three-dimensional structure of chromatin [bib_ref] Loci specific epigenetic drug sensitivity, Zhang [/bib_ref].
DNMTs, HDACs, and histone methyltransferases (HMTs) are among the most widely targeted epigenetic modifiers in contemporary anti-cancer therapies. Inhibitors of DNMTs, HDACs, histone demethylases (HDMs), HMTs and bromodomain proteins are used individually or in combinations to various clinical trials with or without chemotherapy [bib_ref] Bromodomain inhibitor OTX015 in patients with lymphoma or multiple myeloma: a dose-escalation,..., Amorim [/bib_ref] [bib_ref] Epigenetic regulators and their impact on therapy in acute myeloid leukemia, Pastore [/bib_ref] [bib_ref] Targeting epigenetic regulators for cancer therapy: modulation of bromodomain proteins, methyltransferases, demethylases,..., Gelato [/bib_ref]. [fig_ref] Table 1: Various epigenetic modulators in anti-cancer studies and clinical trials [/fig_ref] enlists different types of epigenome-modifying drugs and their utilization, targeting multiple types of solid cancers and hematological malignancies.
## Use of dnmt inhibitors in hindering chemoresistance
DNMT inhibitors are extensively used as tools for hypomethylating the genome in preclinical and clinical studies and the treatment of different types of cancers. Currently, two DNMT inhibitors 5-Azacytidine (AZA, vidaza) and its deoxyribose analog, 5-aza-2 -deoxycytidine (decitabine), are approved by Food and Drug Administration (FDA) as anti-cancer agents. AZA and decitabine are the only epigenetic modulators permitted as therapeutic agents for [bib_ref] Effects of procaine on human nasopharyngeal carcinoma cell strain CNE-2Z, Zhou [/bib_ref] - Nasopharyngeal neoplasms: Phase II (mixture of Procaine with dexamethasone, gentamicin and vitamin B12; unknown status-NCT02735317)
## Zebularine
Inhibits DNMT activity Ovarian cancer [bib_ref] Antimitogenic and chemosensitizing effects of the methylation inhibitor zebularine in ovarian cancer, Balch [/bib_ref] No clinical trial yet
## Hdaci vorinostat/saha inhibits class i and ii hdacs
Leukemia [bib_ref] Activity of histone deacetylase inhibitors and an Aurora kinase inhibitor in BCR-ABL-expressing..., Okabe [/bib_ref] , glioblastoma multiforme (GBM) [bib_ref] Phase I/II trial of vorinostat combined with temozolomide and radiation therapy for..., Galanis [/bib_ref] , breast cancer [bib_ref] The histone deacetylase inhibitor, suberoylanilide hydroxamic acid, overcomes resistance of human breast..., Butler [/bib_ref] [bib_ref] Combination therapy of established cancer using a histone deacetylase inhibitor and a..., Frew [/bib_ref] , melanoma [bib_ref] Histone deacetylase inhibitor sensitizes apoptosis-resistant melanomas to cytotoxic human T lymphocytes through..., Jazirehi [/bib_ref] , pancreatic cancer [bib_ref] The epigenetic agents suberoylanilide hydroxamic acid and 5-AZA-2' deoxycytidine decrease cell proliferation,..., Susanto [/bib_ref] , neuroblastoma [bib_ref] Cooperation of the HDAC inhibitor vorinostat and radiation in metastatic neuroblastoma: efficacy..., Mueller [/bib_ref] , retinoblastoma [bib_ref] Molecular sequelae of histone deacetylase inhibition in human retinoblastoma cell lines: clinical..., Poulaki [/bib_ref] , NSCLC [bib_ref] Vorinostat, SAHA, represses telomerase activity via epigenetic regulation of telomerase reverse transcriptase..., Li [/bib_ref] - [bib_ref] Selective inhibition of BET bromodomains, Filippakopoulos [/bib_ref] , AML [bib_ref] RNAi screen identifies Brd4 as a therapeutic target in acute myeloid leukaemia, Zuber [/bib_ref] , medulloblastoma [bib_ref] BET bromodomain inhibition of MYC-amplified medulloblastoma, Bandopadhayay [/bib_ref] , breast cancer [bib_ref] Response and resistance to BET bromodomain inhibitors in triple-negative breast cancer, Shu [/bib_ref] , and lung cancer [bib_ref] Inhibition of BET bromodomain-dependent XIAP and FLIP expression sensitizes KRAS-mutated NSCLC to..., Klingbeil [/bib_ref] - patients with acute myeloid leukemia (AML) with resistance to conventional chemotherapy. AZA has also been approved for the treatment of chronic myelomonocytic leukemia (CMML) by the European Medicines Agency (EMA) and FDA [bib_ref] DNA methylation inhibitors in cancer: recent and future approaches, Gros [/bib_ref]. Several studies have utilized AZA and decitabine to promote cell cycle arrest and cytotoxicity in resistant cancer cells [bib_ref] Development of gene expression-based score to predict sensitivity of multiple myeloma cells..., Moreaux [/bib_ref] [bib_ref] The role of DNA damage and repair in decitabine-mediated apoptosis in multiple..., Maes [/bib_ref]. In multiple myeloma, AZA reportedly leads to modulation of p16 expression, cleavage of caspase and PARP, and cell cycle arrest at G 0 /G 1 phase [bib_ref] Epigenetic modulating agents as a new therapeutic approach in multiple myeloma, Maes [/bib_ref]. Similarly, decitabine induces G 0 /G 1 and G 2 /M arrest by modulating p21 and p38 [bib_ref] Decitabine induces cell cycle arrest at the G1 phase via p21(WAF1) and..., Lavelle [/bib_ref] ; the effectivity of decitabine at the clinical level has been well established in AML, chronic myelogenous leukemia (CML), and myelodysplasia [bib_ref] Phase 1 study of low-dose prolonged exposure schedules of the hypomethylating agent..., Issa [/bib_ref].
## Clinical trial status
A novel compound, termed SGI-110 (combination of 5-aza-2 -deoxycytidine and guanosine), functions as a prodrug for decitabine and is reported to display promising results against drug-resistant AML and myelodysplasia in Phase III clinical trials (NCT02348489) [bib_ref] S110, a 5-Aza-2'-deoxycytidine-containing dinucleotide, is an effective DNA methylation inhibitor in vivo..., Chuang [/bib_ref] (https://clinicaltrials.gov/). Both AZA and decitabine have undergone many clinical trials before being accepted by FDA for the treatment of several hematological cancers [fig_ref] Table 1: Various epigenetic modulators in anti-cancer studies and clinical trials [/fig_ref]. Currently, clinical trials are going on to elucidate their dosage and potency in treating various solid tumors. Subsequent quests for more efficacious DNMT inhibitors have led to the synthesis of MG98, a second-generation phosphorothioate antisense oligodeoxynucleotide that prevents translation of DNMT1 mRNA. MG98 is currently undergoing phase I/II clinical trials in solid tumors' patients (NCT00003890) [bib_ref] Phase I study of MG98, an oligonucleotide antisense inhibitor of human DNA..., Plummer [/bib_ref]. The clinical trial status of various DNMT inhibitors is listed in [fig_ref] Table 1: Various epigenetic modulators in anti-cancer studies and clinical trials [/fig_ref].
## Hdacis as epi-drugs against chemoresistant cancers
The HDAC family constitutes a major target of the epigenome-modifying drugs as HDAC inhibition can trigger a magnitude of cellular responses, promoting intracellular stress and eventually cell death. The role of HDACs in maintaining chemoresistance has been well established by various studies [bib_ref] SMAR1 repression by pluripotency factors and consequent chemoresistance in breast cancer stem-like..., Bhattacharya [/bib_ref] [bib_ref] HDAC 1/4-mediated silencing of microRNA-200b promotes chemoresistance in human lung adenocarcinoma cells, Chen [/bib_ref] [bib_ref] Dual role of HDAC10 in lysosomal exocytosis and DNA repair promotes neuroblastoma..., Ridinger [/bib_ref] , and the use of HDACis has been in light for several years now. The two HDACis suberoylanilide hydroxamic acid (SAHA, class I and II inhibitor) and depsipeptide (romidepsin, class I inhibitor) have been permitted by FDA for treating T-cell lymphomas [bib_ref] New and emerging HDAC inhibitors for cancer treatment, West [/bib_ref] [bib_ref] Histone deacetylase inhibitor (HDACI) mechanisms of action: emerging insights, Bose [/bib_ref]. SAHA is also reported to induce apoptosis in multiple myelomas through dephosphorylation of Rb, attenuation of Bcl2, and augmentation of p21 and p53 [bib_ref] Molecular sequelae of histone deacetylase inhibition in human malignant B cells, Mitsiades [/bib_ref]. In multiple myeloma, a combination of Panobinostat, a non-selective pan HDACi, and bortezomib is reported to have synergistic activity against Dexamethasone resistance [bib_ref] Treatment of multiple myeloma, Rajkumar [/bib_ref]. The active DNA repair pathway is one of the foremost causes behind chemotherapeutic resistance [bib_ref] DNA damage, repair, and cancer metabolism, Turgeon [/bib_ref]. Studies on various HDACis in different solid and liquid cancers have shown pronounced effects against this active DNA repair system, ultimately hindering the drug resistance ability of the cancer cells. For instance, in AML, a co-treatment with HDACi belinostat and NEDD8-activating enzyme inhibitor pevonedistat reportedly inhibits expression of the DNA repair proteins 53BP1 and RAD51 [bib_ref] The NAE inhibitor pevonedistat interacts with the HDAC inhibitor belinostat to target..., Zhou [/bib_ref]. In neuroblastoma, SAHA is reported to repress DNA repair-mediated acquisition of resistance by inhibiting Ku-86, a significant contributor of non-homologous end-joining DNA damage repair [bib_ref] Cooperation of the HDAC inhibitor vorinostat and radiation in metastatic neuroblastoma: efficacy..., Mueller [/bib_ref]. The pan HDACi valproic acid has been used in clinical trials singly or in combination with other drugs as a tool to target cancer cell resistance. For instance, in neuroendocrine carcinoma and central nervous system (CNS) tumors, treatment with valproic acid displayed significant stability in the patients [bib_ref] Phase 1 study of valproic acid in pediatric patients with refractory solid..., Su [/bib_ref] [bib_ref] A pilot phase II study of valproic acid for treatment of low-grade..., Mohammed [/bib_ref]. Studies reporting valproic acid treatment in combination with various drugs like S-1 (a novel oral fluoropyrimidine derivative), decitabine, bevacizumab have indicated effective anti-tumor activity [bib_ref] Phase I study of anti-VEGF monoclonal antibody bevacizumab and histone deacetylase inhibitor..., Wheler [/bib_ref] [bib_ref] Phase I study of 5-aza-2'-deoxycytidine in combination with valproic acid in non-small-cell..., Chu [/bib_ref] [bib_ref] Effects of valproic acid in combination with S-1 on advanced pancreatobiliary tract..., Iwahashi [/bib_ref].
## Clinical trial status
Compared with DNMTis, HDACis have been studied in several diseases, including hematological malignancies, solid tumors and inflammatory disorders, and have elicited positive outcomes. The three FDA-approved HDACis SAHA, romidepsin, and belinostat, are approved as a therapeutic tool in cutaneous T-cell lymphoma. They are being extensively assessed in the clinical trials for other solid and hematological cancers, either alone or co-treatment with other anti-cancer drugs. For instance, in glioblastoma patients, SAHA is currently under clinical trial combined with radiotherapy and chemotherapeutic drug temozolomide (NCT00731731). A Phase II clinical using romidepsin in peripheral T-cell lymphoma showed an overall 38% response rate [bib_ref] Phase 2 trial of romidepsin in patients with peripheral T-cell lymphoma, Piekarz [/bib_ref]. According to https://clinicaltrials.gov/, approximately 30 clinical trials are ongoing with romidepsin, either as a single agent or combined with other drugs. In Phase II clinical trials, belinostat has been utilized to sensitize platinum-resistant epithelial cancer and micropapillary ovarian tumors [bib_ref] Phase II trial of the histone deacetylase inhibitor belinostat in women with..., Mackay [/bib_ref]. Another phase I/II clinical trial of belinostat in thymic epithelial tumors indicated potency of belinostat-PAC (P: cisplatin, A: adriamycin/doxorubicin C: cyclophosphamide) co-treatment in reducing tumor resistance [bib_ref] A phase I/II trial of belinostat in combination with cisplatin, doxorubicin, and..., Thomas [/bib_ref].
The preclinical and clinical studies using the different HDACis are enlisted in [fig_ref] Table 1: Various epigenetic modulators in anti-cancer studies and clinical trials [/fig_ref].
## Hmt inhibitors in anti-cancer therapeutics
Reversible histone methylation plays a crucial role in the development and progression of various cancers and histone methyltransferase inhibitors (HMTis) are emerging as potential epigenome-modifying tools with promising outcomes in the arena of clinical oncology. Some of the lysine and arginine methyltransferases widely studied in anti-cancer therapeutics include G9a, EZH2, DOT1L (disruptor of telomeric silencing 1-like protein) and PRMT1 (protein arginine methyltransferases 1), inhibitors of which are being extensively investigated [bib_ref] The promise for histone methyltransferase inhibitors for epigenetic therapy in clinical oncology:..., Rugo [/bib_ref]. The expression and activity of EZH2, one of the most well-known HMTs, have been associated with the tumor progression of various cancers [bib_ref] The promise for histone methyltransferase inhibitors for epigenetic therapy in clinical oncology:..., Rugo [/bib_ref]. Enhanced EZH2 activity is reported to induce tumor growth and resistance to conventional chemotherapy, leading to poor prognostic outcomes [bib_ref] Chemosensitive relapse in small cell lung cancer proceeds through an EZH2-SLFN11 axis, Gardner [/bib_ref]. The EZH2 inhibitor tazemetostat, the first FDA-approved HMT inhibitor for cancer treatment, is currently one of the most investigated compounds in the clinical trial, with its anti-cancer properties established in preclinical studies [bib_ref] Preclinical evidence of anti-tumor activity induced by EZH2 inhibition in human models..., Kawano [/bib_ref] [bib_ref] Selective inhibition of EZH2 by EPZ-6438 leads to potent antitumor activity in..., Knutson [/bib_ref]. Other selective inhibitors of EZH2 such as PF-06821497, GSK2816126, are also reported to reduce tumor growth and progression in preclinical models [bib_ref] Optimization of orally bioavailable enhancer of zeste homolog 2 (EZH2) inhibitors using..., Kung [/bib_ref] [bib_ref] EZH2 inhibition as a therapeutic strategy for lymphoma with EZH2-activating mutations, Mccabe [/bib_ref].
EPZ-5676 or pinometostat, a specific inhibitor against H3K79 methyltransferase DOT1L, has been under preclinical investigation for some years and has shown potency in treating the leukemia model [bib_ref] Potent inhibition of DOT1L as treatment of MLL-fusion leukemia, Daigle [/bib_ref].
PRMTs, the HMTs of the arginine methylase family, play a crucial role in cancer signaling pathways. Different classes of PRMTs are reported to be up-regulated and play pro-cancer roles in various solid and hematological cancers [bib_ref] Protein arginine methylation/demethylation and cancer, Poulard [/bib_ref] [bib_ref] Overexpression of CARM1 in breast cancer is correlated with poorly characterized clinicopathologic..., Cheng [/bib_ref] [bib_ref] Aberrant expression of CARM1, a transcriptional coactivator of androgen receptor, in the..., Hong [/bib_ref]. Inhibition of PRMTs has displayed anti-cancer potential in preclinical studies. PF-06939999 and EPZ015666 (GSK3235025), potent orally bioavailable PRMT5 inhibitors, have been reported to show significant antitumor activity [bib_ref] A selective inhibitor of PRMT5 with in vivo and in vitro potency..., Chan-Penebre [/bib_ref].
## Clinical trial status
The EZH2 inhibitors tazemetostat CPI-0209, CPI-1205, GSK2816126, DS-3201, and PF-06821497 have been in clinical trials. Tazemetostat, the most investigated EZH2 inhibitor, has undergone several clinical trials in different types of cancers and has demonstrated potent anti-tumor effects (enlisted in [fig_ref] Table 1: Various epigenetic modulators in anti-cancer studies and clinical trials [/fig_ref]. In patients with refractory malignant mesothelioma with BAP1 inactivation, a Phase II clinical study using tazemetostat has established its activity as an anti-cancer agent with low toxicity (NCT02860286). However, several other clinical trials of tazemetostat in various cancers are still in progress.
EPZ-5676 has been under clinical trials in patients with refractory leukemia (NCT01684150, NCT02141828) and has shown modest clinical activity and an acceptable safety profile [fig_ref] Table 1: Various epigenetic modulators in anti-cancer studies and clinical trials [/fig_ref] [bib_ref] The DOT1L inhibitor pinometostat reduces H3K79 methylation and has modest clinical activity..., Stein [/bib_ref].
Three PRMT5 inhibitors, PF-06939999, GSK3326595 and JNJ-6461978, are currently under clinical trials in patients with solid and hematologic cancers. GSK3368715, the only developed inhibitor of PRMT1, has recently entered Phase I clinical trials in patients with various solid and hematologic malignancies (NCT03666988).
## Bromodomain inhibitors as anti-cancer agents
Bromodomain (BRD) proteins are 'readers' of histone acetylation, which target transcription factors and chromatin-remodeling enzymes to target sites on the chromatin to regulate gene expression. BRD proteins are generally associated with the augmentation of target genes [bib_ref] Bromodomain inhibitors and cancer therapy: from structures to applications, Pérez-Salvia [/bib_ref]. Bromo-and extra-terminal domain (BET) proteins, a crucial subfamily of BRD proteins, constitute BRDT, BRD2, BRD3 and BRD4. Due to their diverse genome-wide targets, BET proteins play significant roles in cellular survival and homeostasis, dysregulation of which leads to malignancy [bib_ref] Achieving clinical success with BET inhibitors as anti-cancer agents, Shorstova [/bib_ref]. Over the recent years, targeting BET proteins through small molecule inhibitors has become a highlight of anti-cancer research to sensitize drug-resistant cancer cells towards therapy. JQ1 and I-BET762 (GSK525762A/molibresib/I-BET) are the two foremost BET inhibitors that have been extensively studied as cancer therapeutics over the past few years. JQ1 has been reported to induce anti-cancer effects in nuclear protein in testis (NUT) midline carcinoma (NMC), AML, medulloblastoma, breast cancer, and lung cancer [bib_ref] Selective inhibition of BET bromodomains, Filippakopoulos [/bib_ref] [bib_ref] RNAi screen identifies Brd4 as a therapeutic target in acute myeloid leukaemia, Zuber [/bib_ref] [bib_ref] BET bromodomain inhibition of MYC-amplified medulloblastoma, Bandopadhayay [/bib_ref] [bib_ref] Response and resistance to BET bromodomain inhibitors in triple-negative breast cancer, Shu [/bib_ref] [bib_ref] Inhibition of BET bromodomain-dependent XIAP and FLIP expression sensitizes KRAS-mutated NSCLC to..., Klingbeil [/bib_ref]. Due to the low oral bioavailability and short half-life of JQ1, it has not been used in clinical trials [bib_ref] Achieving clinical success with BET inhibitors as anti-cancer agents, Shorstova [/bib_ref]. OTX015 (Birabresib), another small molecule similar to JQ1, inhibits BRD2, BRD3 and BRD4 supports oral administration and has been reported to display anti-cancer effects in preclinical studies of drug-resistant neuroblastoma [bib_ref] Targeting MYCN-driven transcription By BET-bromodomain inhibition, Henssen [/bib_ref] , mesothelioma [bib_ref] Promising in vivo efficacy of the BET bromodomain inhibitor OTX015/MK-8628 in malignant..., Vázquez [/bib_ref] , multiple myeloma [bib_ref] Potent activity of the bromodomain inhibitor OTX015 in multiple myeloma, Shi [/bib_ref] , and B-cell lymphoma [bib_ref] The BET bromodomain inhibitor OTX015 affects pathogenetic pathways in preclinical B-cell tumor..., Boi [/bib_ref]. I-BET62 is orally bioavailable and has shown efficacy as an anti-tumor agent in preclinical models, including multiple myeloma [bib_ref] Potent antimyeloma activity of the novel bromodomain inhibitors I-BET151 and I-BET762, Chaidos [/bib_ref] , pancreatic adenocarcinoma [bib_ref] The BET inhibitor I-BET762 inhibits pancreatic ductal adenocarcinoma cell proliferation and enhances..., Xie [/bib_ref] , and neuroblastoma [bib_ref] BET inhibition silences expression of MYCN and BCL2 and induces cytotoxicity in..., Wyce [/bib_ref].
## Clinical trial status
Consecutive research has led to the development of JQ1 analog TEN-010 (JQ2), which has completed Phase I clinical trial in various advanced solid tumors (NCT01987362), AML, and MDS patients (NCT02308761) [bib_ref] A phase 1b dose-escalation/expansion study of BET inhibitor RO6870810 in patients with..., Ramasamy [/bib_ref] [bib_ref] A dose escalation study of RO6870810/TEN-10 in patients with acute myeloid leukemia..., Roboz [/bib_ref] [fig_ref] Table 1: Various epigenetic modulators in anti-cancer studies and clinical trials [/fig_ref].
However, OTX015 ha snot produced any significant positive outcome in clinical trials as the patients administered with OTX015 displayed symptoms of dose-limited toxicities like fatigue and headache, gastrointestinal disorders, anemia, hyperbilirubinemia etc. [bib_ref] Bromodomain inhibitor OTX015 in patients with acute leukaemia: a dose-escalation, phase 1..., Berthon [/bib_ref] [bib_ref] Bromodomain inhibitor OTX015 in patients with lymphoma or multiple myeloma: a dose-escalation,..., Amorim [/bib_ref] [fig_ref] Table 1: Various epigenetic modulators in anti-cancer studies and clinical trials [/fig_ref]. I-BET62 is currently under clinical trial to specify doses [bib_ref] Phase 1 Study of Molibresib (GSK525762), a bromodomain and extra-terminal domain protein..., Piha-Paul [/bib_ref].
## Usage of epi-drugs in targeting cscs
Since CSCs are the major contributors behind drug resistance within the tumor mass, several studies have utilized epi-drugs to specifically target CSCs and sensitize them towards conventional therapies to achieve significant tumor regression [fig_ref] Figure 2: Epidrugs are used to target and sensitize CSCs Several studies have utilized... [/fig_ref]. For instance, DNMT inhibitors AZA and decitabine have been reported to selectively target CSCs of leukemia and bladder cancer, thereby improving the efficacy of chemotherapy [bib_ref] Venetoclax with azacitidine disrupts energy metabolism and targets leukemia stem cells in..., Pollyea [/bib_ref] [bib_ref] Low doses of decitabine improve the chemotherapy efficacy against basal-like bladder cancer..., Wu [/bib_ref]. Inhibition of EZH2 and BET proteins by specific inhibitors have also been reported to repress CSC-associated tumor aggressiveness in different solid cancers [bib_ref] Survival of skin cancer stem cells requires the Ezh2 polycomb group protein, Adhikary [/bib_ref] [bib_ref] The EZH2 inhibitor GSK343 suppresses cancer stem-like phenotypes and reverses mesenchymal transition..., Yu [/bib_ref] [bib_ref] 2020) BET degrader inhibits tumor progression and stem-like cell growth via Wnt/β-catenin..., Tian [/bib_ref]. Similarly, potent HDACis including valproic acid, SAHA and trichostatin A (TSA) have been shown to selectively target CSCs and repress their self-renewal, proliferation, migratory and therapy-resistant properties [bib_ref] Valproic acid suppresses the self-renewal and proliferation of head and neck cancer..., Lee [/bib_ref] [bib_ref] Targeting epigenetic regulation of miR-34a for treatment of pancreatic cancer by inhibition..., Nalls [/bib_ref] [bib_ref] Depletion of HDAC1, 7 and 8 by histone deacetylase inhibition confers elimination..., Cai [/bib_ref]. In a nutshell, all these reports indicate towards the crucial significance of targeting CSCs to alleviate therapy resistance and achieve overall tumor regression.
## Role of epigenetic modulators in anti-tumor immunity
Immunotherapy or enhancing the patient's immune response to eradicate cancer cells has become a breakthrough in anti-cancer therapeutics. But, a substantial percentage of nonresponding patients and systemic toxicities pose an obstacle to its therapeutic success [bib_ref] The contribution of epigenetics to cancer immunotherapy, Villanueva [/bib_ref]. An immunosuppressive tumor milieu can lead to epigenetic modification of tumor-associated immune cells, promoting therapeutic failure [bib_ref] Epigenetic silencing of TH1-type chemokines shapes tumour immunity and immunotherapy, Peng [/bib_ref]. In this context, modulating epigenomic signatures of cancer cells and cancer-associated immune cells show promising prognostic outcomes in patient's responses to immunotherapy.
Epigenetic reprogramming of tumor cells, TME, and tumor-associated immune cells play crucial roles in developing an antitumor immune response, patient's response to immunotherapy, and overall prognostic outcome of the patient. For instance, derepression of endogenous retroviruses (ERVs) by LSD1 inhibitors have been reported to lead to accumulation of double-stranded RNAs (dsRNAs) in cancer cells, which simulates viral infection, in turn triggering antitumoral immunity through interferon response within the tumor milieu [bib_ref] Dual inhibition of DNA and histone methyltransferases increases viral mimicry in ovarian..., Liu [/bib_ref]. Another study has reported that dual inhibition of DNMTs and methyltransferase G9a in ovarian and hematological cancers leads to an augmentation of ERV transcripts and consequent induction of viral defense genes like IRF7 and STAT1 [bib_ref] Reactivation of endogenous retroviral elements via treatment with DNMT-and HDAC-inhibitors, Daskalakis [/bib_ref]. AZA treatment in colorectal cancer has been reported to promote the expression of interferon-response factors like OASL and IRF7 by inducing up-regulation of dsRNA and stimulation of the MDA5/MAVS/IRF7 pathway [bib_ref] DNA-demethylating agents target colorectal cancer cells by inducing viral mimicry by endogenous..., Roulois [/bib_ref]. Inhibition of EZH2 in the ovarian cancer model has been reported to enhance the expression of Th1 chemokine genes (Cxcl9 and Cxcl10), promoting CD8+ T-cell infiltration and leading to the efficacy of anti-PD-L1 antibody immunotherapy [bib_ref] Epigenetic silencing of TH1-type chemokines shapes tumour immunity and immunotherapy, Peng [/bib_ref]. Moreover, due to the crucial role of EZH2 in the differentiation, and maintenance of Treg cells and the expansion of effector T cells, makes it a potential target of immune-modulation therapy [bib_ref] EZH2 is crucial for both differentiation of regulatory T cells and T..., Yang [/bib_ref]. Repression of EZH2 in tumor-infiltrating Treg cells thus promote pro-inflammatory signaling and enhance recruitment of CD8+ and CD4+ effector T cells, ultimately leading to tumor regression [bib_ref] Targeting EZH2 reprograms intratumoral regulatory T cells to enhance cancer immunity, Wang [/bib_ref].
All these reports indicate that targeting epigenetic modulators can prompt significant anti-tumor immunity and thus can serve as a complementary therapeutic approach to current conventional therapies.
## Conclusion and future perspectives
Cancer is the principal cause of global morbidity and mortality, with billions of people succumbing to it each year. The most routinely used therapeutic regimen for most advanced cancers is chemotherapy, which has led to no significant improvement in the treatment scenario and mortality worldwide. Approximately 70-80% of patients with advanced carcinomas experience recurrence, which leads to an even more aggressive form of the disease and is mostly fatal. The development of the recurrent tumor is mainly attributed to drug resistance as the therapy-resistant cells remaining after first-line therapy, serve as the seed for future relapse. Drug resistance involves a complex architecture of molecular events, including heightened drug efflux, drug inactivation, inhibition of apoptosis, and enhanced DNA repair mechanisms. This resistance towards the conventional anti-cancer stratagems, be inherent or acquired, has hindered favorable prognosis since the emergence of the chemotherapy and seems to be a prime impediment in the effective management of breast cancer. Extensive research for the past decade has established that CSCs are the primary drug-resistant cells within the tumor mass, which remains unaffected even after the chemotherapeutic cycles and finally gives rise to recurrent tumors. This indicates the importance of developing CSC targeting therapies that could sensitize the drug-resistant tumor towards conventional chemotherapy and hence will be able to complement it. Combinatorial therapies involving the usage of various drugs or pathway inhibitors and chemotherapy have been highlighted for the past few years due to their efficacy in reducing tumor burden and attenuation of drug resistance.
Epigenetic modifications play a crucial role in the acquisition of therapy resistance. However, even these combinatorial therapies often fall prey to the ingenious epigenetic reprogramming of the cancer cells, leading to treatment failure after a certain point in time. In this scenario, epigenome modifying drugs seem to be an attractive option to serve as a co-treatment procedure along with mainstream/combinatorial therapies. Hence, it is crucial to continue unravelling the intricate epigenetic regulatory mechanisms underlying the acquisition of drug resistance to keep developing more efficacious treatment possibilities. This review encompasses the currently known epigenetic landscape, which regulates the mechanisms of drug resistance in cancer. Further studies are required to enhance overall understanding and devise novel therapeutic strategies to target drug-resistant cancers.
[fig] Figure 1: The epigenetic regulations behind the different mechanisms of drug resistanceSchematic diagram representing the different epigenetic alterations that render increased drug efflux, elevated drug detoxification, augmented DNA repair, and apoptosis resistance properties to the cancer cells, making them resistant towards conventional therapeutic regimens. The interaction between tumor microenvironment and cancer cells, which shapes tumor survival and progression, is also represented schematically. [/fig]
[fig] Figure 2: Epidrugs are used to target and sensitize CSCs Several studies have utilized different epigenetic modulators including DNMT inhibitors, HMT inhibitors, BET inhibitors, and HDACis, to target therapy resistance, self-renewal, proliferation, and migratory potential of CSCs and to sensitize them towards chemotherapy. Abbreviation: AZA, azacytidine; DAC, decitabine; E-Cad, E-cadherin; HMT, histone methyltransferase; N-Cad, N-cadherin; OAA, oxaloacetic acid; SAHA, suberoylanilide hydroxamic acid; TSA, trichostatin A; Vim, vimentin; VPA, valproic acid; α-KG, α-ketoglutarate. [/fig]
[table] Table 1: Various epigenetic modulators in anti-cancer studies and clinical trials [/table]
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Therapeutic Targeting of Autoreactive B Cells: Why, How, and When?
# Introduction: the "why?"
B lymphocytes play a crucial role in development of autoimmunity. In many cases autoantibodies produced by the differentiated daughters of autoreactive B cells mediate disease, such as in systemic lupus erythematosus (SLE), Graves disease, and myasthenia gravis. In other cases autoantibodies, but not B cell function, are dispensable for disease, such as in type 1 diabetes (T1D) and multiple sclerosis (MS). B cells likely participate in disease through non-mutually exclusive ways, including antigen-presentation to T cells, pro-inflammatory cytokine release, and antibody production. Hence, it is not surprising that over the past few decades B cell directed therapies, such as rituximab (anti-CD20), have demonstrated clinical efficacy in treatment of a number of autoimmune diseases.
Nonetheless, pan-B cell depletion therapies, such as rituximab and belimumab (anti-BAFF), are not optimal. These therapeutics are non-specific, targeting both pathogenic and non-pathogenic B cells, resulting in global suppression of humoral immunity. It appears that while pre-existing immunity is spared, development of immunity to newly encountered pathogens, such as SARS-CoV-2, would not. In addition, while B cell depletion typically lasts 6-12 months following cessation of treatment, in some instances individuals never fully recover their B cell compartment. Lastly, anti-CD20 therapies target the majority of B cell subsets but spare antibody producing plasma cells, which do not express CD20 on their cell surface. This fact could explain the varying response to treatment seen in diseases such as SLE and rheumatoid arthritis (RA) in which reduction of autoantibodies is typically incomplete. Hence, attention by pharmaceutical companies and funding agencies has turned to development of antigen-specific therapies that target the pathogenic B cells, ideally including plasma cells, while leaving the remainder of the immune system functioning normally. However, the development of such novel therapeutics has been Biomedicines 2021, 9, 83 2 of 10 hampered by the inherent difficulties in detecting and studying rare antigen-specific B cells that occur at frequencies of <1/100 in the normal B cell repertoire. Here we discuss approaches under development to target autoreactive B cells and the challenges associated with their use in the clinic.
## Methods to target antigen-specific b cells: the "how?"
The basic principle behind antigen-specific targeting of B cells include delivery and binding of antigen and associated toxic cargo to the target cell by virtue of recognition by the antigen receptor (BCR). This requires coupling of the antigen to some form of toxic inhibitory, or apoptosis-inducing molecule or protein, so that when the B cell of interest binds its cognate antigen complex, it is rendered unresponsive or killed. Many different strategies may be employed to accomplish this goal.
Biomedicines 2021, x FOR PEER REVIEW 2 of 10 target the pathogenic B cells, ideally including plasma cells, while leaving the remainder of the immune system functioning normally. However, the development of such novel therapeutics has been hampered by the inherent difficulties in detecting and studying rare antigen-specific B cells that occur at frequencies of <1/100 in the normal B cell repertoire.
Here we discuss approaches under development to target autoreactive B cells and the challenges associated with their use in the clinic.
## Methods to target antigen-specific b cells: the "how?"
The basic principle behind antigen-specific targeting of B cells include delivery and binding of antigen and associated toxic cargo to the target cell by virtue of recognition by the antigen receptor (BCR). This requires coupling of the antigen to some form of toxic inhibitory, or apoptosis-inducing molecule or protein, so that when the B cell of interest binds its cognate antigen complex, it is rendered unresponsive or killed. Many different strategies may be employed to accomplish this goal.
## Particle-based therapeutics
Generation of tolerogenic nanoparticles (tNPs) to treat autoimmunity has recently gained popularity. Synthetic polymer-based nanoparticles have proven safe and offer flexibility in determining the appropriate structure and surface properties, while also allowing efficient carriage of drug and proteins to target cells. In addition, studies have demonstrated tNPs are especially affective at targeting antigen-presenting cells (APCs),
## Particle-based therapeutics
Generation of tolerogenic nanoparticles (tNPs) to treat autoimmunity has recently gained popularity. Synthetic polymer-based nanoparticles have proven safe and offer flexibility in determining the appropriate structure and surface properties, while also allowing efficient carriage of drug and proteins to target cells. In addition, studies have demonstrated tNPs are especially affective at targeting antigen-presenting cells (APCs), such as dendritic cells and macrophages, due to the inherent ability of these APCs to selectively endocytose. For the majority of studies using tNPs to treat autoimmune disease, nanoparticles containing known self-peptides are administered to mice and engulfed by APCs that then present them in the context of MHC to autoreactive T cells in the absence of co-stimulation, resulting in T cell tolerance. However, in order to directly target autoreactive B cells, native autoantigenic epitopes recognized by these cells would need to be displayed on the surface of the nanoparticle. In addition, incorporation of some inhibitory molecule or toxic drug may be required. Multivalent antigen complexes can be very effective at cross-linking and activating B cells compared to soluble antigen, which would result in unwanted activation of self-reactive B cells. However, if properly designed, such complexes can induce B cell unresponsiveness that could be exploited therapeutically.
Macauley et al. utilized liposomal nanoparticles that display both antigen and glycan ligands of CD22, an inhibitory co-receptor on the surface of B cells that when ligated and co-localized with the BCR, induces recruitment of phosphatases that inhibit BCR signaling. Using a mouse model for hemophilia, in which production of anti-Factor VIII (FVIII) antibodies prevents the effective treatment of mice with exogenous FVIII, this group found that these nanoparticles coupled to FVIII, selectively targeted B cells for apoptosis. Deletion of FVIII-reactive B cells resulted in decreased production of anti-FVIII antibodies that allowed successful administration of FVIII to hemophilia mice to prevent bleeding. Similar results were found by another group who used tNPs containing FVIII and rapamycin, an inhibitor of the mTOR pathway and potent immune modulator. While studies using this type of technology have been limited to models of hemophilia, it seems reasonable it could be extended to other models of autoimmunity in the future.
## Soluble antigen arrays
Another method to deliver self-antigen for induction of tolerance has involved soluble multivalent antigen arrays, in which a polymeric backbone is coupled with autoantigen and cell adhesion inhibitory (LABL) peptides. Using this method, Hartwell et al. have shown that co-delivery of the self-antigen PLP with LABL was efficacious in preventing development of experimental autoimmune encephalomyelitis (EAE), a murine model of MS. They demonstrated that these multivalent soluble antigen arrays target antigen-specific B cells directly through binding to the B cell receptor (BCR), rendering the B cell anergic (unresponsive). Using a similar technology to couple insulin to the polymeric background, the same group demonstrated specific binding to insulin-reactive B cells and resulting desensitization. Though it appears promising, it remains to be seen if targeting insulin-reactive B cells using this method results in protection from disease development. Future studies should be aimed at determining the efficacy of multivalent soluble antigen arrays at treating or preventing other forms of autoimmunity.
## Cell-based therapeutics
With the advent of cell-based therapeutics, including generation of chimeric antigen receptor (CAR)-T cells to treat cancer, some researchers have begun to utilize this technology to treat autoimmunity. Generally speaking, CAR-T cells express a chimeric receptor composed of an extracellular domain consisting of a monoclonal antibody fragment that recognizes a specific antigen important in disease pathogenesis. The cytoplasmic portion of the CAR contains signaling domains, most commonly derived from CD28 and CD3ξ, that mediate T cell activation upon antigen recognition. This results in targeted killing or suppression of the cell, depending on whether it is an NK, Teff or Treg cell. In the NOD mouse model of autoimmune diabetes, it is known that presentation of the insulin B chain peptide residues 9-23 in the context of MHCII (I-A g7 ) by APCs to autoreactive T cells is important in development of disease. Zhang et al. generated a monoclonal antibody (mab287) that recognizes the insulin I-A g7 -B:9-23 complex and showed that it effectively blocked recognition by B:9-23 reactive T cells and delayed development of diabetes in 4 of 10 NOD mice. More recently, she generated a 287-CAR CD8+ T cell that targets APCs presenting I-A g7 -B:9-23 and found that it is relatively specific and effective at killing APCs presenting I-A g7 -B:9-23 and delays development of disease. Since antigen presentation by islet-reactive B cells is essential for diabetes development in the NOD, it seems likely that 287-CAR-T cell act by deletion of insulin-reactive B cells. Future studies should be carried out to determine if this is indeed the case.
Other studies have demonstrated specific targeting of autoreactive B cells using CAR-T cell technology. Recently, "BAR" (B-cell-targeting Antibody Receptor) Tregs were shown to prevent anti-FVIII antibody production in the mouse model of hemophilia. In these studies Treg cells bearing chimeric receptors comprised of the immunodominant FVIII epitope linked with the CD28-CD3ξ signaling domains were shown to suppress the anti-FVIII antibody response in vivo in mice by induction of tolerance in FVIII-reactive B cells, and allowed therapeutic administration of FVIII to stop the bleeding. The authors suggested that this effect was mediated by direct FVIIIspecific BAR-Treg suppression of FVIII-reactive B cells and induction of tolerance in T cells important in B cell activation. Similarly, Parvathaneni and Scott generated FVIII-specific BAR-CD8+ T cells, which specifically targeted FVIII-reactive B cells for deletion and reduced anti-FVIII antibody formation in hemophilic mice. These studies demonstrate generation of BAR-Treg and BAR-CD8+ T cells are effective at targeting harmful antibody producing B cells and may prove to be useful in treatment of other autoimmune diseases.
Similarly, Ellebrecht et al. demonstrated chimeric autoantibody receptor (CAAR) T cells could be used to treat pemphigus vulgaris (PV) by selectively depleting desmoglein 3 (Dsg3)-reactive B cells, which are known to mediate disease via autoantibody production. This group transduced human T cells with lentiviral vectors to express a chimeric receptor with an extracellular Dsg3 and intracellular signaling CD137-CD3ξ domains. When administered to a mouse model of PV, the CAAR-T cells depleted circulating Dsg3secreting B cells, reduced anti-Dsg3 IgG levels in serum, and prevented mucosal blistering. More recently, this same group completed a preclinical study of Dsg3-CAART for the treatment of mucosal PV in humans. Their study found Dsg3-CAAR T cells specifically killed human anti-Dsg3 B cells from PV patients ex vivo and showed activity consistent with a threshold dose in vivo. Results from this study has supported an investigational new drug application for a Phase I clinical trial. If approved, results from this study will be an important step toward determining therapeutic potential of BAR/CAAR T cells to treat human autoimmunity.
## Protein and protein fusions
Although recent studies have predominately used particle-based therapeutics, such as nanoparticles or cells to deliver antigen to B cells, soluble protein therapeutics are on the horizon. Benefits of these biologics include their small size, bioavailability, solubility, and increased specificity for targeted B cells. In a proof of concept study, Henry et al. used an anti-insulin monoclonal antibody (mab123) to target insulin-reactive B cells whose BCR is occupied by endogenous insulin using a VH125 heavy chain transgenic NOD mouse model of diabetes. Insulin-reactive B cells were significantly reduced after injection of mab123 in vivo, likely through FcγR mediated depletion. Moreover, since mab123 does not recognize insulin bound to the insulin receptor, mice remained normoglycemic in nondiabetic mice. Importantly, this group demonstrated that systemic administration of mab123 to WT NOD mice resulted in protection from diabetes development.
While this method demonstrates the feasibility of using monoclonal antibodies to target antigen-specific B cells, it requires the BCR of autoreactive B cells to be occupied by antigen. In addition, it would not target B cells that recognize an insulin epitope bound by the 123 antibody, nor immediately adjacent epitopes due to steric hindrance. Moreover, there is concern that administration could result in formation of insulin antibody complexes and ultimately impair the action of physiologic action insulin. Hence to overcome these challenges, Akston Biosciences has created a designer Fc-insulin fusion protein, AKS-107, in which a hormonally inactive insulin is fused to an IgG Fc to specifically target insulin-reactive B cells for complement and FcγR mediated killing or induction of anergy. This approach has the added advantage that the IgG Fc should extend the in vivo half-life of the drug by interaction with FcRN. Promising pre-clinical studies in mice demonstrate AKS-107 induces specific silencing of insulin-reactive B cells and can prevent autoimmune diabetes (unpublished), providing the basis for a first-in-human Phase 1 clinical trial of AKS-107. Results will greatly inform our understanding of the safety and potential applications of autoantigen-IgG Fc fusions to treat T1D, with implications for other autoimmune diseases in which pathogenic antigen-specific B cells are implicated.
While not targeting antigen specific B cells, antibody-based therapies that induce B cell unresponsiveness (anergy) through engagement of FcγRIIb have shown efficacy in treatment of autoimmunity and avoid the harmful side effects of total B cell depletion. FcγRIIb is an inhibitory surface receptor expressed primarily by B cells that when co-engaged with the BCR mediates recruitment of phosphatases that suppress B cell signaling. Xencore Inc developed XmAb5871, which is a humanized mAb that binds to CD19 in the BCR complex and is engineered to have increased affinity for FcγRIIb. Preclinical studies have shown that co-engagement of CD19 and FcγRIIb by XmAb5871 suppresses B cell activation, inhibits upregulation of CD86, and suppresses antibody production. Recently a Phase 2 clinical trial in SLE patients demonstrated that XmAb5871 is well tolerated and significantly suppressed disease recurrence. Another antibody that exploits the inhibitory function of FcγRIIb is the bispecific antibody, PRV-3279 (formally known as MGD010), which binds to CD79b, a signaling component of the BCR, and FcγRIIb. The first-in-human study of MGD010 demonstrated it is tolerated, does not cause B cell depletion but induces downregulation of surface BCR and suppression of BCR-mediated Ca 2+ mobilization, indicative of B cell anergy. Plans to address its potential use in autoimmunity are currently in development. Still, another approach emulates the ability of chronic low avidity antigen exposure, as occurs with soluble autoantigens in vivo, to induce B cell anergy. Antibodies lacking antibody-dependent cell-mediated cytotoxicity (ADCC) and complement fixing ability that are reactive with CD79 induce polyclonal anergy. As shown by the Cambier group and others, these antibodies are effective in mouse models of lupus, T1D, RA and MS (EAE)(Cambier unpublished). While the above candidate therapeutics silence all B cells, the technology could be exploited to generate antibodies that engage the BCR in an antigen-specific manner, similar to AKS-107, which would result in tolerance induction of pathogenic B cells while sparing non-pathogenic B cells.
Another carrier-free antigen-specific therapeutic on the horizon incorporates a drug or toxin directly conjugated to a self-antigen, similar to antibody-drug conjugates (ADCs) currently in use to treat some forms of cancer. In principle, antigen-toxin conjugates would bind specifically to the BCR on pathogenic self-reactive B cells, inducing death by virtue of concentration and cellular uptake of the toxin. In a proof-of-principle study, Pickens et al. demonstrated that conjugation of the self-antigen peptide, PLP 139-152 , to the corticosteroid, dexamethasone, led to protection from development of EAE in mice with increased efficacy compared to dexamethasone treatment alone. While this study utilized a self-antigen fragment and not full-length protein and did not analyze the effects on the immune system in depth, it demonstrates the potential of this type of therapeutic to prevent or delay autoimmunity. Preliminary studies in the Cambier lab have shown that insulin conjugated to the toxin, mitoxantrone, induces specific deletion of murine insulinbinding B cells in vitro (unpublished). Future studies are needed to fully address the therapeutic potential of antigen-toxin conjugates in the treatment of autoimmune disease.
## Challenges to antigen-specific b cell therapy: the "when?"
Despite their infancy, therapies that target antigen-specific B cells for the treatment of autoimmunity show promise and carry an enhanced safety profile relative to current therapies that induce total B cell depletion. However, there are important considerations and potential limitations that should be acknowledged that may limit their clinical efficacy. For example, in autoimmune disorders, such as SLE, many patients develop autoantibodies against more than one antigenand, therefore, simultaneous targeting of B cells with various specificities may be needed for maximal efficacy. On the other hand, while rituximab trials have demonstrated a clear benefit of B cell depletion in MS, knowledge of which autoantigens are important in disease pathogenesis are still not defined. In addition, studies in T1D, MS, RA, and SLE have suggested epitope spreading can occur, such that autoimmune disease could be initiated by one antigen but propagated during chronic autoimmunity by a different antigen, thus making it especially difficult to treat someone with established disease. . Summary of antigen-specific B cell therapies to date.
## Disease model method results references
Hemophilia tNPs coated with FVIII and CD22 ligands FVIII-specific B cell deletiontNP with FVIII encapsulated with rapamycin FVIII-specific B cell deletionFVIII-BAR Treg FVIII-specific B cell tolerance and antibody suppressionFVIII-BAR CD8+ T cell FVIII-specific B cell deletionEAE Soluble antigen array with PLP + LABL PLP-reactive B cell anergyPemphigus Vulgaris Dsg3-CAAR T cell Dsg3-specific B cell deletionAutoimmune diabetes Soluble antigen array with insulin + LABL Insulin-specific B cell anergy ex vivo287-CAR CD8+ T cell I-Ag7-B:9-23 presenting APC deletionmab123
Insulin-binding B cell deletionFc-insulin fusion protein (AKS-107) Insulin-binding B cell deletion/anergy unpublished Insulin-toxin Insulin-binding B cell deletion unpublished Importantly, proper timing of therapy may be crucial. In T1D, RA, and SLE, patients can develop autoantibodies many years before onset of disease and, therefore, targeting of antigen-specific B cells once disease is confirmed may be too late. Conversely, many healthy individuals generate autoantibodies but never have autoimmune disease, and therefore, preemptive treatment based on autoantibody positivity may be unnecessary for some individuals. Other aspects to consider include the potential need for multimodal therapies that target pathogenic B cells as well as T cells and/or cytokines for the most effective treatment. Moreover, while cell-based antigen-specific therapies, such as BAR and CAAR T cells, have proven to be beneficial, the time and cost of ex vivo manipulation of autologous patient cells may limit their translational potential to treat human autoimmunity.
Ultimately, the largest theoretical impediment to the success of therapies that specifically target autoreactive B cells is the fact that by the time the patient reaches the clinic, and thus the first opportunity for therapeutic intervention, they are producing high levels of autoantibody. These autoantibodies can be expected to bind and neutralize the candidate autoantigen therapeutics before they reach their B cell target. Utility of autoreactive B cellspecific therapies may be relegated to situations in which genetic risk dictates a certainty that disease will develop, yet autoantibodies are not yet being produced.
# Conclusions
Despite many concerns and challenges, therapeutic silencing of autoreactive B cells remains a very attractive prospect. Thus, it is an exciting time for the development of antigen-specific therapeutics to treat autoimmunity. Current and future studies will inform our understanding of the therapeutic efficacy of these treatments to prevent, manage, and/or cure autoimmune disease. |
Polyetheretherketone and Its Composites for Bone Replacement and Regeneration
In this article, recent advances in the development, preparation, biocompatibility and mechanical properties of polyetheretherketone (PEEK) and its composites for hard and soft tissue engineering are reviewed. PEEK has been widely employed for fabricating spinal fusions due to its radiolucency, chemical stability and superior sterilization resistance at high temperatures. PEEK can also be tailored into patient-specific implants for treating orbital and craniofacial defects in combination with additive manufacturing process. However, PEEK is bioinert, lacking osseointegration after implantation. Accordingly, several approaches including surface roughening, thin film coating technology, and addition of bioactive hydroxyapatite (HA) micro-/nanofillers have been adopted to improve osseointegration performance. The elastic modulus of PEEK is 3.7-4.0 GPa, being considerably lower than that of human cortical bone ranging from 7-30 GPa. Thus, PEEK is not stiff enough to sustain applied stress in load-bearing orthopedic implants. Therefore, HA micro-/nanofillers, continuous and discontinuous carbon fibers are incorporated into PEEK for enhancing its stiffness for load-bearing applications. Among these, carbon fibers are more effective than HA micro-/nanofillers in providing additional stiffness and load-bearing capabilities. In particular, the tensile properties of PEEK composite with 30wt% short carbon fibers resemble those of cortical bone. Hydrophobic PEEK shows no degradation behavior, thus hampering its use for making porous bone scaffolds. PEEK can be blended with hydrophilic polymers such as polyglycolic acid and polyvinyl alcohol to produce biodegradable scaffolds for bone tissue engineering applications. Polymers 2020, 12, 2858 2 of 48 fabrication of materials, microstructural and mechanical characterizations, in vitro cellular tests and in vivo animal models, and final clinical trials (Figure 1) [2]. Polymers 2020, 8, x 2 of 51 biomaterials for bone tissue engineering applications involve several stage procedures including proper selection and fabrication of materials, microstructural and mechanical characterizations, in vitro cellular tests and in vivo animal models, and final clinical trials (Figure 1) [2].Figure 1. Multiple stages involved in the assessment of newly developed biomaterials for bone tissue engineering. Reproduced from [2] with permission of MDPI under the conditions of Creative Commons Attribution license.Biomaterials used in bone tissue engineering generally include ceramics, metals, polymers and composites. Bioceramics like calcium phosphates are well known bioactive materials that mimic and integrate with human bones by inducing an apatite layer on their surfaces[3,4]. However, brittle CaPbioceramics exhibit low fracture toughness, thereby confining their use as coating materials for metallic implants. Metallic materials with excellent mechanical strength and ductility, e.g., titanium alloy, cobalt chrome molybdenum alloy and 316L stainless steel are commonly used for making loadbearing bone replacements, dental implants, spinal fusion and bone fixation devices[5][6][7]. However, metallic implants have certain drawbacks including stress shielding effect and corrosion issues. The former derives from a significant mismatch in elastic modulus between the implant and host bone, leading to the reduction of bone mass and implant loosening[8]. The elastic modulus, tensile strength and elongation at fracture of human cortical bone range from 7-30 GPa, 50-150 MPa and 1-3%, respectively[9]. The elastic moduli of CoCrMo alloy, austenitic stainless steel and Ti-6Al-4V alloy are reported to be 200 and 110 GPa, respectively [10]. So, the modulus of metallic alloys is remarkably higher than that of cortical bone, resulting in stress shielding and implant failure. Corrosion is due to the release of metal ions through the interaction of implants with body fluid. In particular, released Ni 2+ , Cr 3+ and Co 2+ ions from the CoCrMo alloy and 316L stainless steel would cause allergy, inflammation and cytotoxicity[11,12]. Moreover, chloride ions in human body fluid induce pitting corrosion on those alloys due to the breakdown of passive films[11,13,14].Polymers with facile processability, good chemical resistance and light weight are attractive materials for orthopedic applications[15][16][17][18][19][20]. Biodegradable polymers with poor mechanical strength such as polylactic acid, polyglycolic acid, and their copolymers are generally employed for making scaffolds for tissue engineering applications[15,16]. The biocompatibility and mechanical performance of biodegradable polymers can be enhanced by adding bioactive nanofillers[21][22][23]. Non-degradable polymers such as polyethylene and polyetheretherketone (PEEK) find applications in orthopedics where long-term stability is needed. High-density polyethylene (HDPE) is typically used for tendon repair and catheter tube, while ultra-high molecular weight polyethylene Polymers 2020, 12, 2858 3 of 48 as a bearing material in total joint prostheses[24]. HDPE is very ductile with an elastic modulus of 1.3 GPa[25]. Its mechanical strength and stiffness can be improved by reinforcing with hydroxyapatite (HA) having a stiffness of 120.6 GPa[26]. Hydroxyapatite [Ca 10 (PO 4 ) 6 (OH) 2 ] is a bioceramic having compositional similarity with the mineral component of human bones and teeth. Human bone tissue is a biocomposite consisting of 70% nano-hydroxyapatite (nHA) platelets and 30% collagen fibrils. To mimic the structural component of human bones, HAPEX TM composite has been developed for bone replacement by incorporating 40vol% HA into the HDPE matrix[25]. However, Young's modulus of HAPEX TM (4.4 GPa) is considerably lower than that of cortical bone, rendering its application for non-load bearing maxillofacial implants only.PEEK is a semicrystalline polymer with an aromatic backbone chain connecting by the ketone and ether groups. PEEK is considered as a high-performance polymer due to its excellent chemical resistance, high melting temperature (340 - C), superior radiation and sterilization resistance, high modulus of elasticity (3.7-4.0 GPa) and tensile strength (103 MPa). Moreover, PEEK is a radiolucent material, facilitating radiographic assessment of the implants[27,28]. In this respect, PEEK has been successfully used as a biomaterial for spinal fusion, cranial reconstruction and dental implant applications[28][29][30][31][32][33][34][35][36][37][38]. In spite of these advantages, however, bioinert PEEK is unfavorable for osteoblastic cell adhesion. As such, HA microparticles, HA whiskers and other ceramic particulates are incorporated into PEEK for enhancing bone cell adhesion and mechanical stiffness[39][40][41]. For example, Abu Bakar et al. added 5-40 vol% HA microparticles (mHA) with a mean size of 25.68 µm to PEEK. In vivo pig model revealed that 20 vol% mHA fillers promote bone regeneration for PEEK. However, the tensile strength of PEEK decreased markedly from 80 MPa to 58 and 44 MPa by adding 20 and 40 vol% mHA, respectively[39]. Thus, the composites had lower tensile strength than pure PEEK, i.e., the fillers carried no applied load during tensile testing. This was attributed to the fracture of large mHA fillers and poor interfacial mHA-PEEK bonding. To enhance the stiffness and mechanical strength of PEEK, continuous and discontinuous carbon fibers (CFs) with high elastic modulus were added to PEEK accordingly[42]. The PEEK/CF composites have been used for lumbar interbody fusion cages, acetabular cup of hip prostheses, and dental implants/abutments[43][44][45][46][47].In recent years, advances in nanotechnology have greatly contributed to the creation of novel nanomaterials with functional properties for biomedical applications[48,49]. Nanomaterials with small size and large surface area exhibit superior chemical, mechanical and physical properties compared to their micron-sized system[50][51][52][53]. As an example, nanohydroxyapatite (nHA) has shown improved bone cell adhesion over mHA[54][55][56][57][58][59]. Thus, nHA is incorporated into both degradable and non-degradable polymers for enhancing osteoblastic adhesion and proliferation[60][61][62][63][64][65][66][67][68][69]. Apart from nHA, carbonaceous nanofillers of different dimensions such as carbon nanotubes (CNTs), carbon nanofibers (CNFs) and graphene oxide sheets also promote the adhesion, growth and differentiation of osteoblasts[70][71][72][73][74][75][76][77][78][79][80][81]. As a result, carbon-based nanomaterials are also incorporated into polymers for enhancing their biocompatibility in many tissue engineering applications[61][62][63][82][83][84][85][86]. Recently, Ma et al. fabricated PEEK/nHA nanocomposites by means of vibrating ball-mill mixing and injection molding[87]. By adding 40 wt % (21.5 vol%) nHA, Young's modulus of PEEK/nHA reaches 4.6 GPa, being considerably smaller than the lower limit of the modulus of cortical bone, i.e., 7 GPa. Tjong and coworkers melt-compounded PEEK/nHA nanocomposites in an extruder followed by injection molding. The Young's modulus of PEEK/21.5 vol% nHA nanocomposite exceeds that of cortical bone, i.e., 7.85 GPa[62]. However, this material is extremely brittle with a low tensile elongation of 0.63%. To tackle the issue of brittleness, low loading levels of CNFs or CNTs are added to PEEK/nHA nanocomposites[62,63]. This article gives a comprehensive and updated review of studies relating the development, fabrication, biocompatibility and mechanical properties of PEEK and its composites for hard and soft bone tissue engineering applications. Polymers 2020, 12, 2858 4 of 48PEEK ImplantsPEEK has been widely employed as spinal implant materials including cages, rods, and screws to maintain rigid stability in the spine such that the bones can fuse together gradually[28][29][30][31]88,89]. PEEK spinal fusions can prevent serious problems of stress shielding and vertebral collapse typically encountered by metallic materials. PEEK can also be used to fabricate patient specific implants (PSIs) for reconstructing maxillofacial deformities[31,34,[90][91][92]. PEEK PSIs demonstrate higher clinical efficacy in restoring the shape of the damaged orbit[90]. Recently, additive manufacturing (AM) has emerged as an effective tool for printing complex objects for biomedical applications[93][94][95]. AM technology is particularly useful to make patient-specific cranial implants using a computer aided design (CAD)/computer aided manufacturing (CAM) software. In the process, the skull of a patient is scanned by computer tomography, and the data are delivered to a CAD/CAM system for manufacturing three-dimensional (3D) PEEK PSIs(Figure 2). Reliable clinical results for PEEK PSIs in maxillofacial surgery have been reported by Jarvinen et al. very recently [91]. Meanwhile, Bagul et al. and Serra et al. have printed 3D PEEK lumbar cages using fused filament fabrication (FFF), or generally known as fused deposition modeling (FDM). FFF is one of the AM techniques which employs polymer filaments created by the hot melt extrusion for printing 3D medical implants layer-by-layer. The 3D printed cages are capable of providing mechanical strength for lumbar cage applications [94]. Polymers 2020, 8, x 4 of 51PEEK ImplantsPEEK has been widely employed as spinal implant materials including cages, rods, and screws to maintain rigid stability in the spine such that the bones can fuse together gradually[28][29][30][31]88,89]. PEEK spinal fusions can prevent serious problems of stress shielding and vertebral collapse typically encountered by metallic materials. PEEK can also be used to fabricate patient specific implants (PSIs) for reconstructing maxillofacial deformities[31,34,[90][91][92]. PEEK PSIs demonstrate higher clinical efficacy in restoring the shape of the damaged orbit[90]. Recently, additive manufacturing (AM) has emerged as an effective tool for printing complex objects for biomedical applications[93][94][95]. AM technology is particularly useful to make patient-specific cranial implants using a computer aided design (CAD)/computer aided manufacturing (CAM) software. In the process, the skull of a patient is scanned by computer tomography, and the data are delivered to a CAD/CAM system for manufacturing three-dimensional (3D) PEEK PSIs(Figure 2). Reliable clinical results for PEEK PSIs in maxillofacial surgery have been reported by Jarvinen et al. very recently [91]. Meanwhile, Bagul et al. and Serra et al. have printed 3D PEEK lumbar cages using fused filament fabrication (FFF), or generally known as fused deposition modeling (FDM). FFF is one of the AM techniques which employs polymer filaments created by the hot melt extrusion for printing 3D medical implants layerby-layer. The 3D printed cages are capable of providing mechanical strength for lumbar cage applications [94].
# Introduction
Bone fractures and segmental bone defects pose a significant risk of patient morbidity and economic burden to the public healthcare system [bib_ref] Management of segmental bone defects, Mauffrey [/bib_ref]. With increasing numbers of geriatric population and people suffering from traffic and sports injuries, bone cancer and disorders, dental defects and congenital disorders, the demands for hip replacement, craniomaxillofacial and spinal implants are rising globally in recent years. In this perspective, the development of novel biomaterials for hard tissue replacements is considered of technological importance. Biomaterials for medical devices should be non-toxic to cells, having remarkable biocompatibility and bioactivity for the regeneration of bone tissues. Osseointegration of medical implants with the surrounding tissues plays a decisive role for new bone regeneration and healing in maxillofacial reconstruction, spinal fusion and hip replacement applications. In general, the development and successful implementation of novel biomaterials for bone tissue engineering applications involve several stage procedures including proper selection and Biomaterials used in bone tissue engineering generally include ceramics, metals, polymers and composites. Bioceramics like calcium phosphates are well known bioactive materials that mimic and integrate with human bones by inducing an apatite layer on their surfaces [bib_ref] Calcium phosphate bioceramics: A review of their history, structure, properties, coating technologies..., Eliaz [/bib_ref] [bib_ref] Biological properties of calcium phosphate biomaterials for bone repair: A review, Lu [/bib_ref]. However, brittle CaP-bioceramics exhibit low fracture toughness, thereby confining their use as coating materials for metallic implants. Metallic materials with excellent mechanical strength and ductility, e.g., titanium alloy, cobalt chrome molybdenum alloy and 316L stainless steel are commonly used for making load-bearing bone replacements, dental implants, spinal fusion and bone fixation devices [bib_ref] The biological response to orthopaedic implants for joint replacement: Part I: Metals, Gibon [/bib_ref] [bib_ref] Metallic biomaterials: Current challenges and opportunities, Prasad [/bib_ref]. However, metallic implants have certain drawbacks including stress shielding effect and corrosion issues. The former derives from a significant mismatch in elastic modulus between the implant and host bone, leading to the reduction of bone mass and implant loosening [bib_ref] Elastic modulus in the selection of interbody implants, Heary [/bib_ref]. The elastic modulus, tensile strength and elongation at fracture of human cortical bone range from 7-30 GPa, 50-150 MPa and 1-3%, respectively [bib_ref] Hydrostatically extruded HAPEX TM, Wang [/bib_ref]. The elastic moduli of CoCrMo alloy, austenitic stainless steel and Ti-6Al-4V alloy are reported to be [bib_ref] Preparation, characterization, cellular response and in vivo osseointegration of polyetheretherketone/nano-hydroxyapatite/carbon fiber ternary..., Deng [/bib_ref] and 110 GPa, respectively [bib_ref] Fundamentals of medical implant materials, Nag [/bib_ref]. So, the modulus of metallic alloys is remarkably higher than that of cortical bone, resulting in stress shielding and implant failure. Corrosion is due to the release of metal ions through the interaction of implants with body fluid. In particular, released Ni 2+ , Cr 3+ and Co 2+ ions from the CoCrMo alloy and 316L stainless steel would cause allergy, inflammation and cytotoxicity [bib_ref] Comparison of metal release from various metallic biomaterials in vitro, Okazaki [/bib_ref] [bib_ref] Molecular analysis of chromium and cobalt-related toxicity, Scharf [/bib_ref]. Moreover, chloride ions in human body fluid induce pitting corrosion on those alloys due to the breakdown of passive films [bib_ref] Comparison of metal release from various metallic biomaterials in vitro, Okazaki [/bib_ref] [bib_ref] Electron and ion spectroscopic studies of the passive film on iron-chromium alloys, Tjong [/bib_ref] [bib_ref] ESCA and SIMS studies of the passive film on iron, Tjong [/bib_ref].
Polymers with facile processability, good chemical resistance and light weight are attractive materials for orthopedic applications [bib_ref] Biomedical applications of biodegradable polymers, Ulery [/bib_ref] [bib_ref] Synthetic polymers for biomedical applications, Wei [/bib_ref] [bib_ref] Polymeric biomaterials for bone regeneration, Shi [/bib_ref] [bib_ref] Synthesis and proton conductivities of phosphonic acid containing poly-(arylene ether)s, Meng [/bib_ref] [bib_ref] Synthesis and degradation behavior of poly (propylene carbonate) derived from carbon dioxide..., Du [/bib_ref] [bib_ref] Rheology and morphology of compatibilized polyamide 6 blends containing liquid crystalline copolyesters, Meng [/bib_ref]. Biodegradable polymers with poor mechanical strength such as polylactic acid, polyglycolic acid, and their copolymers are generally employed for making scaffolds for tissue engineering applications [bib_ref] Biomedical applications of biodegradable polymers, Ulery [/bib_ref] [bib_ref] Synthetic polymers for biomedical applications, Wei [/bib_ref]. The biocompatibility and mechanical performance of biodegradable polymers can be enhanced by adding bioactive nanofillers [bib_ref] Antibacterial activities of aliphatic polyester nanocomposites with silver nanoparticles and/or graphene oxide..., Liao [/bib_ref] [bib_ref] Melt-compounded polylactic acid composite hybrids with hydroxyapatite nanorods and silver nanoparticles: Biodegradation,..., Liu [/bib_ref]. Non-degradable polymers such as polyethylene and polyetheretherketone (PEEK) find applications in orthopedics where long-term stability is needed. High-density polyethylene (HDPE) is typically used for tendon repair and catheter tube, while ultra-high molecular weight polyethylene (UHMWPE) is employed In general, poor osseointegration at the bone-implant interface would lead to a failure of the medical devices. The biological interface between a device and the surrounding host tissue plays a key role in the overall performance of implants. Clinical strategies to increase osseointegration and osteoinduction of orthopedic implants still remain a big challenge to surgeons, chemists and materials scientists. PEEK-based implants with smooth and hydrophobic surfaces are bioinert, having limited capacity to bind to bone tissues. As a result, several approaches such as the creation of porous feature, surface modification and coating deposition can be used to enhance osseointegration ability of PEEK [bib_ref] Impaction durability of porous polyether-ether-ketone (PEEK) and titanium-coated PEEK interbody fusion devices, Torstrick [/bib_ref] [bib_ref] Recent advances in the reconstruction of cranio-maxillofacial defects using computer-aided design/computer-aided manufacturing, Oh [/bib_ref] [bib_ref] Clinical efficacy of peek patient-specific implants in orbital reconstruction, Chepurnyi [/bib_ref] [bib_ref] Do surface porosity and pore size influence mechanical properties and cellular response..., Torstrick [/bib_ref] [bib_ref] Getting PEEK to stick to bone: The development of porous PEEK for..., Torstrick [/bib_ref] [bib_ref] Porous PEEK improves the bone-implant interface compared to plasma-sprayed titanium coating on..., Torstrick [/bib_ref] [bib_ref] In vivo experimental study of anterior cervical fusion using bioactive polyetheretherketone in..., Shimizu [/bib_ref] [bib_ref] Role of implants surface modification in osseointegration: A systematic review, Liu [/bib_ref] [bib_ref] Current strategies to improve the bioactivity of PEEK, Ma [/bib_ref] [bib_ref] Nano-hydroxyapatite-coated PEEK implants: A pilot study in rabbit bone, Barkarmo [/bib_ref] [bib_ref] Polyether ether ketone implants achieve increased bone fusion when coated with nano-sized..., Johanson [/bib_ref] [bib_ref] Nanosized hydroxyapatite coating on PEEK implants enhances early bone formation: A histological..., Johanson [/bib_ref] [bib_ref] Cold-spray coating of hydroxyapatite on a three-dimensional polyetheretherketone implant and its biocompatibility..., Lee [/bib_ref]. Porous features offer a remarkable osteogenic In general, poor osseointegration at the bone-implant interface would lead to a failure of the medical devices. The biological interface between a device and the surrounding host tissue plays a key role in the overall performance of implants. Clinical strategies to increase osseointegration and osteoinduction of orthopedic implants still remain a big challenge to surgeons, chemists and materials scientists. PEEK-based implants with smooth and hydrophobic surfaces are bioinert, having limited capacity to bind to bone tissues. As a result, several approaches such as the creation of porous feature, surface modification and coating deposition can be used to enhance osseointegration ability of PEEK [bib_ref] Impaction durability of porous polyether-ether-ketone (PEEK) and titanium-coated PEEK interbody fusion devices, Torstrick [/bib_ref] [bib_ref] Recent advances in the reconstruction of cranio-maxillofacial defects using computer-aided design/computer-aided manufacturing, Oh [/bib_ref] [bib_ref] Clinical efficacy of peek patient-specific implants in orbital reconstruction, Chepurnyi [/bib_ref] [bib_ref] Do surface porosity and pore size influence mechanical properties and cellular response..., Torstrick [/bib_ref] [bib_ref] Getting PEEK to stick to bone: The development of porous PEEK for..., Torstrick [/bib_ref] [bib_ref] Porous PEEK improves the bone-implant interface compared to plasma-sprayed titanium coating on..., Torstrick [/bib_ref] [bib_ref] In vivo experimental study of anterior cervical fusion using bioactive polyetheretherketone in..., Shimizu [/bib_ref] [bib_ref] Role of implants surface modification in osseointegration: A systematic review, Liu [/bib_ref] [bib_ref] Current strategies to improve the bioactivity of PEEK, Ma [/bib_ref] [bib_ref] Nano-hydroxyapatite-coated PEEK implants: A pilot study in rabbit bone, Barkarmo [/bib_ref] [bib_ref] Polyether ether ketone implants achieve increased bone fusion when coated with nano-sized..., Johanson [/bib_ref] [bib_ref] Nanosized hydroxyapatite coating on PEEK implants enhances early bone formation: A histological..., Johanson [/bib_ref] [bib_ref] Cold-spray coating of hydroxyapatite on a three-dimensional polyetheretherketone implant and its biocompatibility..., Lee [/bib_ref]. Porous features offer a remarkable osteogenic response at the cell level, thereby facilitating bone ingrowth of spinal fixation devices. However, the presence of pores throughout entire PEEK implants would degrade the mechanical strength greatly. In this respect, Torstrick et al. have introduced a porous surface layer of few micrometers thick with high interconnectivity on PEEK using a melt extrusion and porogen (NaCl) leaching process. The porous surface layer facilitates mechanical interlocking of bone, without sacrificing mechanical performance of PEEK. Porous PEEK with a surface porosity of 67.3% and a mean pore size of 279.9 µm retains 73.9% of the strength and 73.4% of the elastic modulus of solid PEEK [bib_ref] The use of patient specific polyetheretherketone implants for reconstruction of maxillofacial deformities, Jarvinen [/bib_ref]. Moreover, surface porous PEEK facilitates cell attachment and proliferation of human femoral osteoblasts (hOBs) and human mesenchymal stem cells (hMSCs) [bib_ref] Do surface porosity and pore size influence mechanical properties and cellular response..., Torstrick [/bib_ref] [bib_ref] Getting PEEK to stick to bone: The development of porous PEEK for..., Torstrick [/bib_ref]. The surface porous structure promotes vascularization and bone ingrowth at 6 and 12 weeks after implantation into a rat femoral segmental defect model [bib_ref] The use of patient specific polyetheretherketone implants for reconstruction of maxillofacial deformities, Jarvinen [/bib_ref]. On the basis of mechanical characterization, in vitro cellular response and in vivo animal model results, PEEK interbody fusion implant with a solid core and porous structure on the superior and inferior faces has been developed by Vertera Spine (Atlanta, GA, USA) under the tradename COHERE ® . COHERE ® shows a successful clinical use in anterior cervical discectomy and fusion (ACDF) surgery and becomes a popular choice of biomaterial for cervical myelopathy treatment [bib_ref] Getting PEEK to stick to bone: The development of porous PEEK for..., Torstrick [/bib_ref].
## Coatings
Deposition of PEEK surface with osteoconductive materials like HA, Ti and titanium dioxide (TiO 2 ) can enhance its bioactivity and osseointegration greatly [bib_ref] In vivo experimental study of anterior cervical fusion using bioactive polyetheretherketone in..., Shimizu [/bib_ref] [bib_ref] Role of implants surface modification in osseointegration: A systematic review, Liu [/bib_ref] [bib_ref] Current strategies to improve the bioactivity of PEEK, Ma [/bib_ref] [bib_ref] Nano-hydroxyapatite-coated PEEK implants: A pilot study in rabbit bone, Barkarmo [/bib_ref] [bib_ref] Polyether ether ketone implants achieve increased bone fusion when coated with nano-sized..., Johanson [/bib_ref] [bib_ref] Nanosized hydroxyapatite coating on PEEK implants enhances early bone formation: A histological..., Johanson [/bib_ref] [bib_ref] Cold-spray coating of hydroxyapatite on a three-dimensional polyetheretherketone implant and its biocompatibility..., Lee [/bib_ref]. Those surface coatings can be deposited on PEEK using several techniques including plasma immersion ion implantation (PIII), ionic plasma deposition (IPD), vacuum plasma spraying (VPS), arc ion plating (AIP), cold spraying, spin coating, aerosol deposition, radio-frequency (RF), and magnetron sputtering [fig_ref] Figure 3: Current strategies typically used to improve bioactivity of PEEK [/fig_ref]. Among these, plasma spraying can yield rough and porous coating on PEEK, which is beneficial for bone cell adhesion and ingrowth. The technique involves the use of an arc inside the plasma gun to melt ceramic or metal powders at high temperatures. The molten droplets are then propelled onto the target substrate, followed by flattening, rapid solidification, and cooling to form a porous coating layer [fig_ref] Figure 4: Schematic illustration of air plasma spray set-up [/fig_ref]. The spraying process can be performed in a vacuum chamber instead of air at atmospheric pressure to prevent the oxidation of the coating material, which is referred to as 'vacuum plasma spraying'.
Polymers 2020, response at the cell level, thereby facilitating bone ingrowth of spinal fixation devices. However, the presence of pores throughout entire PEEK implants would degrade the mechanical strength greatly. In this respect, Torstrick et al. have introduced a porous surface layer of few micrometers thick with high interconnectivity on PEEK using a melt extrusion and porogen (NaCl) leaching process. The porous surface layer facilitates mechanical interlocking of bone, without sacrificing mechanical performance of PEEK. Porous PEEK with a surface porosity of 67.3% and a mean pore size of 279.9 µm retains 73.9% of the strength and 73.4% of the elastic modulus of solid PEEK [bib_ref] The use of patient specific polyetheretherketone implants for reconstruction of maxillofacial deformities, Jarvinen [/bib_ref]. Moreover, surface porous PEEK facilitates cell attachment and proliferation of human femoral osteoblasts (hOBs) and human mesenchymal stem cells (hMSCs) [bib_ref] Do surface porosity and pore size influence mechanical properties and cellular response..., Torstrick [/bib_ref] [bib_ref] Getting PEEK to stick to bone: The development of porous PEEK for..., Torstrick [/bib_ref]. The surface porous structure promotes vascularization and bone ingrowth at 6 and 12 weeks after implantation into a rat femoral segmental defect model [bib_ref] The use of patient specific polyetheretherketone implants for reconstruction of maxillofacial deformities, Jarvinen [/bib_ref]. On the basis of mechanical characterization, in vitro cellular response and in vivo animal model results, PEEK interbody fusion implant with a solid core and porous structure on the superior and inferior faces has been developed by Vertera Spine (Atlanta, GA, USA) under the tradename COHERE ® . COHERE ® shows a successful clinical use in anterior cervical discectomy and fusion (ACDF) surgery and becomes a popular choice of biomaterial for cervical myelopathy treatment [bib_ref] Getting PEEK to stick to bone: The development of porous PEEK for..., Torstrick [/bib_ref].
## Coatings
Deposition of PEEK surface with osteoconductive materials like HA, Ti and titanium dioxide (TiO2) can enhance its bioactivity and osseointegration greatly [bib_ref] In vivo experimental study of anterior cervical fusion using bioactive polyetheretherketone in..., Shimizu [/bib_ref] [bib_ref] Role of implants surface modification in osseointegration: A systematic review, Liu [/bib_ref] [bib_ref] Current strategies to improve the bioactivity of PEEK, Ma [/bib_ref] [bib_ref] Nano-hydroxyapatite-coated PEEK implants: A pilot study in rabbit bone, Barkarmo [/bib_ref] [bib_ref] Polyether ether ketone implants achieve increased bone fusion when coated with nano-sized..., Johanson [/bib_ref] [bib_ref] Nanosized hydroxyapatite coating on PEEK implants enhances early bone formation: A histological..., Johanson [/bib_ref] [bib_ref] Cold-spray coating of hydroxyapatite on a three-dimensional polyetheretherketone implant and its biocompatibility..., Lee [/bib_ref]. Those surface coatings can be deposited on PEEK using several techniques including plasma immersion ion implantation (PIII), ionic plasma deposition (IPD), vacuum plasma spraying (VPS), arc ion plating (AIP), cold spraying, spin coating, aerosol deposition, radio-frequency (RF), and magnetron sputtering [fig_ref] Figure 3: Current strategies typically used to improve bioactivity of PEEK [/fig_ref]. Among these, plasma spraying can yield rough and porous coating on PEEK, which is beneficial for bone cell adhesion and ingrowth. The technique involves the use of an arc inside the plasma gun to melt ceramic or metal powders at high temperatures. The molten droplets are then propelled onto the target substrate, followed by flattening, rapid solidification, and cooling to form a porous coating layer [fig_ref] Figure 4: Schematic illustration of air plasma spray set-up [/fig_ref]. The spraying process can be performed in a vacuum chamber instead of air at atmospheric pressure to prevent the oxidation of the coating material, which is referred to as 'vacuum plasma spraying'.
## Hydroxyapatite-coated peek implants
Vacuum plasma spraying is capable of forming rough and porous bioceramic coatings on PEEK for promoting in-growth of bone cells into the coatings. The process is carried out at high temperatures, typically at about 2000 °C or above for melting HA with a melting temperature of 1670 °C. However, inhomogeneous melting of HA into tricalcium phosphate (Ca3(PO4)2) and tetracalcium phosphate (Ca4O(PO4)2) during plasma spraying would deteriorate the performance of deposited coatings [bib_ref] Plasma-sprayed Hydroxyapatite-based coatings: Chemical, mechanical, microstructural, and biomedical properties, Heimann [/bib_ref]. To prevent nonuniform HA formation and thermal damage of polymer substrate under high temperature of plasma, cold spraying can be employed to deposit HA layer on PEEK at lower temperatures. The cold spray process utilizes the energy stored in high pressure compressed gas to accelerate powder particles at very high velocities, ca. 300-1200 m/s. The particles with high kinetic energy impact on the target without melting, forming a homogenous surface coating accordingly. For instance, Lee et al. coated HA on PEEK using cold spraying process. The HA-PEEK samples promoted the differentiation of bone marrow-derived mesenchymal stem cells as manifested by enhanced alkaline phosphatase activity, calcium production, and bone sialoprotein (BSP) expression [bib_ref] Cold-spray coating of hydroxyapatite on a three-dimensional polyetheretherketone implant and its biocompatibility..., Lee [/bib_ref]. Furthermore, the insertion of HA-PEEK cage implant into minipig model showed the regeneration of bone tissues.
Alternatively, aerosol deposition can also effectively deposit ceramic powders on the polymer substrates to form coatings at lower temperatures. Aerosol deposition is a spray coating process using an aerosol generator in which the carrier gas and ceramic powders are contained in a vibrating chamber. The aerosolized particles are then transported to the deposition chamber with a slit nozzle to impact them on the target material at velocities between 100 and 600 m/s [fig_ref] Figure 5: Schematic illustration of typical coating formation using aerosol deposition [/fig_ref] [bib_ref] Moos, R. The aerosol deposition method: A modified aerosol generation unit to..., Hanft [/bib_ref]. Hahn et al. coated PEEK with an HA layer by means of aerosol deposition followed by hydrothermal annealing for enhancing the crystallinity of HA [bib_ref] Osteoconductive hydroxyapatite coated PEEK for spinal fusion surgery, Hahn [/bib_ref]. As a result, HA-PEEK exhibited excellent biocompatibility under in vitro cell culture and in vivo animal model tests. Rabiei and Sandukas deposited HA on PEEK using RF magnetron sputtering. Prior to HA deposition, an yttria-stabilized zirconia (YSZ) layer was first coated on the PEEK substrate to provide heat shielding. The asdeposited HA layer exhibited an amorphous structure. Then HA/YSZ coated PEEK was subjected to microwave annealing in order to form crystalline HA [bib_ref] Processing and evaluation of bioactive coatings on polymeric implants, Rabiei [/bib_ref]. Cell cultivation tests revealed a marked increase in initial bone cell attachment and proliferation on microwave-annealed samples, compared with uncoated PEEK and PEEK with amorphous HA surface layer. In another study, Rabiei and coworkers used ion beam assisted deposition (IBAD) to form HA/YSZ coatings on cylindrical PEEK implants. Thereafter, microwave heating with and without subsequent autoclaving were employed to crystallize amorphous HA layer [bib_ref] Hydroxyapatite coating on PEEK implants: Biomechanical and histological study in a rabbit..., Durham [/bib_ref]. The HA/YSZ-PEEK samples were implanted into lateral femoral condyle of the rabbits. The heat-treated HA/YSZ-PEEK samples were found to exhibit
## Hydroxyapatite-coated peek implants
Vacuum plasma spraying is capable of forming rough and porous bioceramic coatings on PEEK for promoting in-growth of bone cells into the coatings. The process is carried out at high temperatures, typically at about 2000 - C or above for melting HA with a melting temperature of 1670 - C. However, inhomogeneous melting of HA into tricalcium phosphate (Ca 3 (PO 4 ) 2 ) and tetracalcium phosphate (Ca 4 O(PO 4 ) 2 ) during plasma spraying would deteriorate the performance of deposited coatings [bib_ref] Plasma-sprayed Hydroxyapatite-based coatings: Chemical, mechanical, microstructural, and biomedical properties, Heimann [/bib_ref]. To prevent nonuniform HA formation and thermal damage of polymer substrate under high temperature of plasma, cold spraying can be employed to deposit HA layer on PEEK at lower temperatures. The cold spray process utilizes the energy stored in high pressure compressed gas to accelerate powder particles at very high velocities, ca. 300-1200 m/s. The particles with high kinetic energy impact on the target without melting, forming a homogenous surface coating accordingly. For instance, Lee et al. coated HA on PEEK using cold spraying process. The HA-PEEK samples promoted the differentiation of bone marrow-derived mesenchymal stem cells as manifested by enhanced alkaline phosphatase activity, calcium production, and bone sialoprotein (BSP) expression [bib_ref] Cold-spray coating of hydroxyapatite on a three-dimensional polyetheretherketone implant and its biocompatibility..., Lee [/bib_ref]. Furthermore, the insertion of HA-PEEK cage implant into minipig model showed the regeneration of bone tissues.
Alternatively, aerosol deposition can also effectively deposit ceramic powders on the polymer substrates to form coatings at lower temperatures. Aerosol deposition is a spray coating process using an aerosol generator in which the carrier gas and ceramic powders are contained in a vibrating chamber. The aerosolized particles are then transported to the deposition chamber with a slit nozzle to impact them on the target material at velocities between 100 and 600 m/s [fig_ref] Figure 5: Schematic illustration of typical coating formation using aerosol deposition [/fig_ref] [bib_ref] Moos, R. The aerosol deposition method: A modified aerosol generation unit to..., Hanft [/bib_ref]. Hahn et al. coated PEEK with an HA layer by means of aerosol deposition followed by hydrothermal annealing for enhancing the crystallinity of HA [bib_ref] Osteoconductive hydroxyapatite coated PEEK for spinal fusion surgery, Hahn [/bib_ref]. As a result, HA-PEEK exhibited excellent biocompatibility under in vitro cell culture and in vivo animal model tests. Rabiei and Sandukas deposited HA on PEEK using RF magnetron sputtering. Prior to HA deposition, an yttria-stabilized zirconia (YSZ) layer was first coated on the PEEK substrate to provide heat shielding. The as-deposited HA layer exhibited an amorphous structure. Then HA/YSZ coated PEEK was subjected to microwave annealing in order to form crystalline HA [bib_ref] Processing and evaluation of bioactive coatings on polymeric implants, Rabiei [/bib_ref]. Cell cultivation tests revealed a marked increase in initial bone cell attachment and proliferation on microwave-annealed samples, compared with uncoated PEEK and PEEK with amorphous HA surface layer. In another study, Rabiei and coworkers used ion beam assisted deposition (IBAD) to form HA/YSZ coatings on cylindrical PEEK implants. Thereafter, microwave heating with and without subsequent autoclaving were employed to crystallize amorphous HA layer [bib_ref] Hydroxyapatite coating on PEEK implants: Biomechanical and histological study in a rabbit..., Durham [/bib_ref]. The HA/YSZ-PEEK samples were implanted into lateral femoral condyle of improved osseointegration, higher bone regeneration and bone-implant contact area when compared to pure PEEK. Recently, Johansson et al. reported that spin-coated nHA-PEEK screws exhibit a remarkable improvement in osteogenic properties. The nHA-PEEK screws were implanted into the tibia and femur of rabbits for 3-12 weeks [fig_ref] Figure 6: Histologic section of nHA-coated PEEK screw implanted into the rabbit tibia for... [/fig_ref] [bib_ref] Polyether ether ketone implants achieve increased bone fusion when coated with nano-sized..., Johanson [/bib_ref] [bib_ref] Nanosized hydroxyapatite coating on PEEK implants enhances early bone formation: A histological..., Johanson [/bib_ref]. The nHA-PEEK after 3 weeks showed osseointegration as revealed by higher bone implant contact (BIC) and total bone area percentage values compared to uncoated PEEK. After 12 weeks of implantation, BIC was significant enhanced in femur and tibia around HA-PEEK screws compared with pure PEEK [bib_ref] Polyether ether ketone implants achieve increased bone fusion when coated with nano-sized..., Johanson [/bib_ref] [bib_ref] Nanosized hydroxyapatite coating on PEEK implants enhances early bone formation: A histological..., Johanson [/bib_ref].
## Ti-coated peek implants
Hydrophobic PEEK surface often reduces cellular adhesion and growth. Thus, implant surfaces with adequate hydrophilicity favor cell adhesion and enhance interactions with surrounding tissues. In this perspective, PEEK can be coated with a titanium surface layer for improving its wettability, resulting in better cell attachment and biocompatibility [bib_ref] The electron beam deposition of titanium on polyetheretherketone (PEEK) and the resulting..., Han [/bib_ref] [bib_ref] Porous titanium-coated polyetheretherketone implants exhibit an improved bone-implant interface: An in vitro..., Cheng [/bib_ref] [bib_ref] Evaluating osseointegration into a deeply porous titanium scaffold, Guyer [/bib_ref] [bib_ref] Plasma sprayed titanium coating to PEEK improves the bone-implant interface, Walsh [/bib_ref]. For instance, Han et al. coated Ti onto PEEK using electron beam deposition. The Ti layer adhered firmly to the PEEK substrate and Recently, Johansson et al. reported that spin-coated nHA-PEEK screws exhibit a remarkable improvement in osteogenic properties. The nHA-PEEK screws were implanted into the tibia and femur of rabbits for 3-12 weeks [fig_ref] Figure 6: Histologic section of nHA-coated PEEK screw implanted into the rabbit tibia for... [/fig_ref] [bib_ref] Polyether ether ketone implants achieve increased bone fusion when coated with nano-sized..., Johanson [/bib_ref] [bib_ref] Nanosized hydroxyapatite coating on PEEK implants enhances early bone formation: A histological..., Johanson [/bib_ref]. The nHA-PEEK after 3 weeks showed osseointegration as revealed by higher bone implant contact (BIC) and total bone area percentage values compared to uncoated PEEK. After 12 weeks of implantation, BIC was significant enhanced in femur and tibia around HA-PEEK screws compared with pure PEEK [bib_ref] Polyether ether ketone implants achieve increased bone fusion when coated with nano-sized..., Johanson [/bib_ref] [bib_ref] Nanosized hydroxyapatite coating on PEEK implants enhances early bone formation: A histological..., Johanson [/bib_ref]. Recently, Johansson et al. reported that spin-coated nHA-PEEK screws exhibit a remarkable improvement in osteogenic properties. The nHA-PEEK screws were implanted into the tibia and femur of rabbits for 3-12 weeks [fig_ref] Figure 6: Histologic section of nHA-coated PEEK screw implanted into the rabbit tibia for... [/fig_ref] [bib_ref] Polyether ether ketone implants achieve increased bone fusion when coated with nano-sized..., Johanson [/bib_ref] [bib_ref] Nanosized hydroxyapatite coating on PEEK implants enhances early bone formation: A histological..., Johanson [/bib_ref]. The nHA-PEEK after 3 weeks showed osseointegration as revealed by higher bone implant contact (BIC) and total bone area percentage values compared to uncoated PEEK. After 12 weeks of implantation, BIC was significant enhanced in femur and tibia around HA-PEEK screws compared with pure PEEK [bib_ref] Polyether ether ketone implants achieve increased bone fusion when coated with nano-sized..., Johanson [/bib_ref] [bib_ref] Nanosized hydroxyapatite coating on PEEK implants enhances early bone formation: A histological..., Johanson [/bib_ref].
## Ti-coated peek implants
Hydrophobic PEEK surface often reduces cellular adhesion and growth. Thus, implant surfaces with adequate hydrophilicity favor cell adhesion and enhance interactions with surrounding tissues. In this perspective, PEEK can be coated with a titanium surface layer for improving its wettability, resulting in better cell attachment and biocompatibility [bib_ref] The electron beam deposition of titanium on polyetheretherketone (PEEK) and the resulting..., Han [/bib_ref] [bib_ref] Porous titanium-coated polyetheretherketone implants exhibit an improved bone-implant interface: An in vitro..., Cheng [/bib_ref] [bib_ref] Evaluating osseointegration into a deeply porous titanium scaffold, Guyer [/bib_ref] [bib_ref] Plasma sprayed titanium coating to PEEK improves the bone-implant interface, Walsh [/bib_ref]. For instance, Han et al. coated Ti onto PEEK using electron beam deposition. The Ti layer adhered firmly to the PEEK substrate and Reprinted from [bib_ref] Nanosized hydroxyapatite coating on PEEK implants enhances early bone formation: A histological..., Johanson [/bib_ref] with permission of MDPI under the terms and conditions of the Creative Commons Attribution license.
## Ti-coated peek implants
Hydrophobic PEEK surface often reduces cellular adhesion and growth. Thus, implant surfaces with adequate hydrophilicity favor cell adhesion and enhance interactions with surrounding tissues. In this perspective, PEEK can be coated with a titanium surface layer for improving its wettability, resulting in better cell attachment and biocompatibility [bib_ref] The electron beam deposition of titanium on polyetheretherketone (PEEK) and the resulting..., Han [/bib_ref] [bib_ref] Porous titanium-coated polyetheretherketone implants exhibit an improved bone-implant interface: An in vitro..., Cheng [/bib_ref] [bib_ref] Evaluating osseointegration into a deeply porous titanium scaffold, Guyer [/bib_ref] [bib_ref] Plasma sprayed titanium coating to PEEK improves the bone-implant interface, Walsh [/bib_ref] onto PEEK using electron beam deposition. The Ti layer adhered firmly to the PEEK substrate and improved its wettability [bib_ref] The electron beam deposition of titanium on polyetheretherketone (PEEK) and the resulting..., Han [/bib_ref]. As such, modified PEEK surface promoted the adhesion of mouse osteoblastic cell line (MC3T3-E1). Moreover, in vivo animal tests revealed that Ti-PEEK implants have a higher BIC ratio than pure PEEK implants.
Very recently, Cheng et al. studied in vitro cell culture and in vivo sheep model of Ti-PEEK disks prepared by vacuum plasma spraying [bib_ref] Porous titanium-coated polyetheretherketone implants exhibit an improved bone-implant interface: An in vitro..., Cheng [/bib_ref]. The plasma sprayed Ti coating is rough and porous [fig_ref] Figure 7: Scanning electron microscopic images showing [/fig_ref]. Rough Ti-PEEK disks induce the adhesion and proliferation of osteoblasts and upregulate the gene expressions including alkaline phosphatase (ALP) and bone morphogenetic protein-2 (BMP-2) [bib_ref] Osteoblasts exhibit a more differentiated phenotype and increased bone morphogenetic protein production..., Olivares-Navarrete [/bib_ref]. ALP activity is considered as an early marker of cell differentiation; BMP-2is the growth factor that promotes osteoblastic cell differentiation [bib_ref] Osteoblast differentiation at a glance, Rutkovskiy [/bib_ref] [bib_ref] Bone morphogenetic proteins for nucleus pulposus regeneration, Krouwels [/bib_ref]. From the literature, human osteoblast-like osteosarcoma cells (MG63) cultured on the surfaces with increasing roughness show decreased proliferation and increased cell differentiation [bib_ref] Implant surface characteristics modulate differentiation behavior of cells in the osteoblastic lineage, Schwartz [/bib_ref]. Cheng et al. also reported a reduced proliferation, but high ALP and BMP-2 levels of MG63 on Ti-PEEK [fig_ref] Figure 8: BMP-2 protein levels secreted by MG63 cells cultivated on Ti-PEEK, PEEK, and... [/fig_ref]. In vivo animal tests showed that Ti-PEEK samples exhibit enhanced osseointegration and increased bone formation compared to unmodified PEEK. Biomechanical pullout measurement revealed that Ti-PEEK samples have higher pullout force after 12 and 24 weeks of implantation. The higher pullout strength derived from mechanical interlocking between the implant and bone. Similarly, Walsh et al. demonstrated that the titanium coating significantly enhanced the shear strength at the bone-implant interface of plasma sprayed Ti-PEEK after 4 weeks of implantation into cortical bone of adult sheep. Furthermore, the shear strength of Ti-PEEK continued to increase with time compared to PEEK [bib_ref] Plasma sprayed titanium coating to PEEK improves the bone-implant interface, Walsh [/bib_ref]. improved its wettability [bib_ref] The electron beam deposition of titanium on polyetheretherketone (PEEK) and the resulting..., Han [/bib_ref]. As such, modified PEEK surface promoted the adhesion of mouse osteoblastic cell line (MC3T3-E1). Moreover, in vivo animal tests revealed that Ti-PEEK implants have a higher BIC ratio than pure PEEK implants. Very recently, Cheng et al. studied in vitro cell culture and in vivo sheep model of Ti-PEEK disks prepared by vacuum plasma spraying [bib_ref] Porous titanium-coated polyetheretherketone implants exhibit an improved bone-implant interface: An in vitro..., Cheng [/bib_ref]. The plasma sprayed Ti coating is rough and porous [fig_ref] Figure 7: Scanning electron microscopic images showing [/fig_ref]. Rough Ti-PEEK disks induce the adhesion and proliferation of osteoblasts and upregulate the gene expressions including alkaline phosphatase (ALP) and bone morphogenetic protein-2 (BMP-2) [bib_ref] Osteoblasts exhibit a more differentiated phenotype and increased bone morphogenetic protein production..., Olivares-Navarrete [/bib_ref]. ALP activity is considered as an early marker of cell differentiation; BMP-2is the growth factor that promotes osteoblastic cell differentiation [bib_ref] Osteoblast differentiation at a glance, Rutkovskiy [/bib_ref] [bib_ref] Bone morphogenetic proteins for nucleus pulposus regeneration, Krouwels [/bib_ref]. From the literature, human osteoblast-like osteosarcoma cells (MG63) cultured on the surfaces with increasing roughness show decreased proliferation and increased cell differentiation [bib_ref] Implant surface characteristics modulate differentiation behavior of cells in the osteoblastic lineage, Schwartz [/bib_ref]. Cheng et al. also reported a reduced proliferation, but high ALP and BMP-2 levels of MG63 on Ti-PEEK [fig_ref] Figure 8: BMP-2 protein levels secreted by MG63 cells cultivated on Ti-PEEK, PEEK, and... [/fig_ref]. In vivo animal tests showed that Ti-PEEK samples exhibit enhanced osseointegration and increased bone formation compared to unmodified PEEK. Biomechanical pullout measurement revealed that Ti-PEEK samples have higher pullout force after 12 and 24 weeks of implantation. The higher pullout strength derived from mechanical interlocking between the implant and bone. Similarly, Walsh et al. demonstrated that the titanium coating significantly enhanced the shear strength at the bone-implant interface of plasma sprayed Ti-PEEK after 4 weeks of implantation into cortical bone of adult sheep. Furthermore, the shear strength of Ti-PEEK continued to increase with time compared to PEEK [bib_ref] Plasma sprayed titanium coating to PEEK improves the bone-implant interface, Walsh [/bib_ref].
It is noted that titanium coating deposited on PEEK is susceptible to wear and delamination [bib_ref] Does impaction of titanium-coated interbody fusion cages into the disc space cause..., Kienle [/bib_ref]. The coating delamination would induce implant loosening, while the wear debris of metal particles could lead to chronic inflammation. Wear debris have raised concerns about the safety of Ti-PEEK implants. Nevertheless, Ti-PEEK cages have been used for posterior lumbar interbody fusion (PLIF) surgery very recently. Makino et al. employed computed tomography (CT) color mapping to assess bone ongrowth on the Ti-PEEK cages in 24 patients experiencing PLIF. Porous Ti-PEEK cages used were PROSPACE ® XP (Aesculap AG, Tuttlingen, Germany) [bib_ref] Computed tomography color mapping for evaluation of bone ongrowth on the surface..., Makino [/bib_ref]. A total of 248 surfaces of cage frames were assessed from the patients at six months after surgery. Bone ongrowth was observed in 134 of 248 images of interbody cages (54.0%) by CT color mapping [bib_ref] Computed tomography color mapping for evaluation of bone ongrowth on the surface..., Makino [/bib_ref].
## Tio2-coated peek implants
Titanium dioxide (titania; TiO2) exhibits good bioactivity and photocatalytic antibacterial properties [bib_ref] Titanium oxide antibacterial surfaces in biomedical devices, Visai [/bib_ref] [bib_ref] Using photocatalyst metal oxides as antimicrobial surface coatings to ensure food safety-Opportunities..., Yemmireddy [/bib_ref]. Titania has been deposited on stainless steel pins of fixation devices, showing the ability to reduce the pin site infection [bib_ref] Clinical and histomorphometrical study on titanium dioxide-coated external fixation pins, Koseki [/bib_ref]. So TiO2 layer can be deposited on PEEK for enhancing its osteogenic and antibacterial activities. Titania exists three polymorphs including anatase, rutile and brookite. Titania can react readily with water molecules in aqueous environments, forming hydroxyl groups on its surface. The hydroxyl groups of TiO2 provide active sites for depositing bone-like calcium phosphate (apatite) in a simulated body fluid (SBF). The calcium and phosphate ions can bind to the hydroxylated TiO2 surface, facilitating osseointegration accordingly [bib_ref] Molecular investigation of the initial nucleation of calcium phosphate on TiO 2..., Zheng [/bib_ref].
Recently, Tsou et al. deposited TiO2 layer on PEEK using arc ion plating (AIP) for spinal implant applications. Anatase-TiO2 (A-TiO2) and rutile-TiO2 (R-TiO2) coatings were achieved by controlling the target current and substrate bias voltage during deposition. The coated samples were implanted It is noted that titanium coating deposited on PEEK is susceptible to wear and delamination [bib_ref] Does impaction of titanium-coated interbody fusion cages into the disc space cause..., Kienle [/bib_ref]. The coating delamination would induce implant loosening, while the wear debris of metal particles could lead to chronic inflammation. Wear debris have raised concerns about the safety of Ti-PEEK implants. Nevertheless, Ti-PEEK cages have been used for posterior lumbar interbody fusion (PLIF) surgery very recently. Makino et al. employed computed tomography (CT) color mapping to assess bone ongrowth on the Ti-PEEK cages in 24 patients experiencing PLIF. Porous Ti-PEEK cages used were PROSPACE ® XP (Aesculap AG, Tuttlingen, Germany) [bib_ref] Computed tomography color mapping for evaluation of bone ongrowth on the surface..., Makino [/bib_ref]. A total of 248 surfaces of cage frames were assessed from the patients at six months after surgery. Bone ongrowth was observed in 134 of 248 images of interbody cages (54.0%) by CT color mapping [bib_ref] Computed tomography color mapping for evaluation of bone ongrowth on the surface..., Makino [/bib_ref].
## Tio 2 -coated peek implants
Titanium dioxide (titania; TiO 2 ) exhibits good bioactivity and photocatalytic antibacterial properties [bib_ref] Titanium oxide antibacterial surfaces in biomedical devices, Visai [/bib_ref] [bib_ref] Using photocatalyst metal oxides as antimicrobial surface coatings to ensure food safety-Opportunities..., Yemmireddy [/bib_ref]. Titania has been deposited on stainless steel pins of fixation devices, showing the ability to reduce the pin site infection [bib_ref] Clinical and histomorphometrical study on titanium dioxide-coated external fixation pins, Koseki [/bib_ref]. So TiO 2 layer can be deposited on PEEK for enhancing its osteogenic and antibacterial activities. Titania exists three polymorphs including anatase, rutile and brookite. Titania can react readily with water molecules in aqueous environments, forming hydroxyl groups on its surface. The hydroxyl groups of TiO 2 provide active sites for depositing bone-like calcium phosphate (apatite) in a simulated body fluid (SBF). The calcium and phosphate ions can bind to the hydroxylated TiO 2 surface, facilitating osseointegration accordingly [bib_ref] Molecular investigation of the initial nucleation of calcium phosphate on TiO 2..., Zheng [/bib_ref].
Recently, Tsou et al. deposited TiO 2 layer on PEEK using arc ion plating (AIP) for spinal implant applications. Anatase-TiO 2 (A-TiO 2 ) and rutile-TiO 2 (R-TiO 2 ) coatings were achieved by controlling the target current and substrate bias voltage during deposition. The coated samples were implanted into the femurs of New Zealand white male rabbits. The shear stress needed for disrupting the bone-implant interface at different time points was determined by the push-out test [bib_ref] In vivo osseointegration performance of titanium dioxide coating modified polyetheretherketone using arc..., Tsou [/bib_ref]. The R-TiO 2 /PEEK implant exhibits a shear strength of 6.51 MPa at 12 weeks of implantation, being 2.5 times higher than the shear stress of pure PEEK [fig_ref] Figure 9: Shear strength between the bone tissue and implant for pure PEEK, A-TiO2/PEEK... [/fig_ref]. The R-TiO 2 /PEEK implant shows superior osseointegration and fixation than A-TiO 2 /PEEK due to the abundance of negatively charged hydroxyl groups on its surface [bib_ref] In vivo osseointegration performance of titanium dioxide coating modified polyetheretherketone using arc..., Tsou [/bib_ref]. Shimizu et al. employed the sol-gel technique to deposit a thin TiO 2 layer on PEEK [bib_ref] In vivo experimental study of anterior cervical fusion using bioactive polyetheretherketone in..., Shimizu [/bib_ref] [bib_ref] Bioactivity of sol-gel-derived TiO 2 coating on polyetheretherketone: In vitro and in..., Shimizu [/bib_ref]. The sol-gel method is advantageous over plasma spraying and AIP due to its low processing temperature, thus without damaging the structure of PEEK. They demonstrated that TiO 2 -PEEK surfaces favor the formation of apatite upon soaking in SBF. Those surface modified PEEK implants exhibit better bone-bonding properties than uncoated PEEK on the basis of in vivo biomechanical and histological examinations of rabbit tibia model [bib_ref] Bioactivity of sol-gel-derived TiO 2 coating on polyetheretherketone: In vitro and in..., Shimizu [/bib_ref]. Despite these benefits, TiO 2 -PEEK implants are not readily available for clinical applications.
higher than the shear stress of pure PEEK [fig_ref] Figure 9: Shear strength between the bone tissue and implant for pure PEEK, A-TiO2/PEEK... [/fig_ref]. The R-TiO2/PEEK implant shows superior osseointegration and fixation than A-TiO2/PEEK due to the abundance of negatively charged hydroxyl groups on its surface [bib_ref] In vivo osseointegration performance of titanium dioxide coating modified polyetheretherketone using arc..., Tsou [/bib_ref]. Shimizu et al. employed the sol-gel technique to deposit a thin TiO2 layer on PEEK [bib_ref] In vivo experimental study of anterior cervical fusion using bioactive polyetheretherketone in..., Shimizu [/bib_ref] [bib_ref] Bioactivity of sol-gel-derived TiO 2 coating on polyetheretherketone: In vitro and in..., Shimizu [/bib_ref]. The sol-gel method is advantageous over plasma spraying and AIP due to its low processing temperature, thus without damaging the structure of PEEK. They demonstrated that TiO2-PEEK surfaces favor the formation of apatite upon soaking in SBF. Those surface modified PEEK implants exhibit better bone-bonding properties than uncoated PEEK on the basis of in vivo biomechanical and histological examinations of rabbit tibia model [bib_ref] Bioactivity of sol-gel-derived TiO 2 coating on polyetheretherketone: In vitro and in..., Shimizu [/bib_ref]. Despite these benefits, TiO2-PEEK implants are not readily available for clinical applications.
## Peek-based microcomposites
## Peek/ha microcomposites
The mechanical strength and stiffness of polymers can be enhanced by adding reinforcing materials of micro/nano scale, forming the so-called "microcomposites" and "nanocomposites". As mentioned, the modulus of elasticity of pure PEEK is 3.7-4.0 GPa [bib_ref] High-performance nanocomposites based on polyetherketones, Díez-Pascual [/bib_ref] [bib_ref] PEEK biomaterials in trauma, orthopedic, and spinal implants, Kurtz [/bib_ref] , being lower than that of human cortical bone ranging from 7 to 30 GPa [bib_ref] Hydrostatically extruded HAPEX TM, Wang [/bib_ref]. Therefore, many efforts have been spent by the researchers in enhancing the stiffness and strength of PEEK by reinforcing with HA microparticles, HA whiskers and carbon fibers for load-bearing orthopedic applications [bib_ref] Tensile properties, tension-tension fatigue and biological response of polyetheretherketone-hydroxyapatite composites for load-bearing..., Bakar [/bib_ref] [bib_ref] Processing and tensile properties of hydroxyapatite-whisker-reinforced polyetheretherketone, Converse [/bib_ref] [bib_ref] The advantages of BioHPP polymer as superstructure material in oral implantology, Bechir [/bib_ref] [bib_ref] Investigation of mechanical impact behavior of short carbon-fiber-reinforced PEEK composites, Garcia-Gonzalez [/bib_ref] [bib_ref] First results of a new vacuum plasma sprayed (VPS) titanium-coated carbon/PEEK composite..., Hoppe [/bib_ref] [bib_ref] Suitability of carbon fiber-reinforced polyetheretherketone cages for use as anterior struts following..., Heary [/bib_ref] [bib_ref] Carbon fiber reinforced PEEK composites based on 3D-printing technology for orthopedic and..., Han [/bib_ref] [bib_ref] Carbon fibre/polyether ether ketone (CF/PEEK) implants in orthopaedic oncology, Laux [/bib_ref] [bib_ref] Use of carbon-fiber-reinforced composite implants in orthopedic surgery, Hak [/bib_ref] [bib_ref] Tension-tension fatigue behavior of hydroxyapatite reinforced polyetheretherketone composites, Tang [/bib_ref] [bib_ref] Mechanical properties and in vitro response of strontium-containing hydroxyapatite/polyetheretherketone composites, Wong [/bib_ref] [bib_ref] Does PEEK/HA enhance bone formation compared with PEEK in a sheep cervical..., Walsh [/bib_ref] [bib_ref] Formulation of a covalently bonded hydroxyapatite and poly (ether ether ketone) composite, Hughes [/bib_ref] [bib_ref] Evaluating the bioactivity of a hydroxyapatite-incorporated polyetheretherketone biocomposite, Ma [/bib_ref] [bib_ref] The influence of sterilization processes on the micromechanical properties of carbon fiber-reinforced..., Godora [/bib_ref] [bib_ref] Carbon fiber reinforced PEEK optima-A composite material biomechanical properties and wear/debris characteristics..., Steinberg [/bib_ref]. The elastic modulus of HA is 120.6 GPa [bib_ref] Ab initio elastic properties and tensile strength of crystalline hydroxyapatite, Ching [/bib_ref] , so it can be used as a reinforcement to stiffen PEEK. Abu Bakar et al. incorporated 5-40 vol% mHA into PEEK, resulting in microcomposites with improved stiffness [bib_ref] Tensile properties, tension-tension fatigue and biological response of polyetheretherketone-hydroxyapatite composites for load-bearing..., Bakar [/bib_ref] [bib_ref] Tension-tension fatigue behavior of hydroxyapatite reinforced polyetheretherketone composites, Tang [/bib_ref]. However, the tensile strength of PEEK-mHA microcomposites was smaller than that of pure PEEK. The tensile strength of PEEK decreased markedly from 78.30 MPa to 64.71 and 43.76 MPa by adding 10 vol% and 40 vol% mHA, respectively. The reduction in tensile strength of PEEK-mHA microcomposites was attributed to a poor mHA-PEEK interfacial bonding, leading to decohesion of mHA particles from the polymer matrix. Moreover, the inclusion of 40 vol% mHA into a ductile PEEK matrix led to brittle fracture [fig_ref] Figure 1: Multiple stages involved in the assessment of newly developed biomaterials for bone... [/fig_ref]. The final failure strain of PEEK reduced markedly from 28.93% to 19.23% and 0.98%, respectively by adding 10 vol% and 40 vol% mHA.
## Peek-based microcomposites
## Peek/ha microcomposites
The mechanical strength and stiffness of polymers can be enhanced by adding reinforcing materials of micro/nano scale, forming the so-called "microcomposites" and "nanocomposites". As mentioned, the modulus of elasticity of pure PEEK is 3.7-4.0 GPa [bib_ref] High-performance nanocomposites based on polyetherketones, Díez-Pascual [/bib_ref] [bib_ref] PEEK biomaterials in trauma, orthopedic, and spinal implants, Kurtz [/bib_ref] , being lower than that of human cortical bone ranging from 7 to 30 GPa [bib_ref] Hydrostatically extruded HAPEX TM, Wang [/bib_ref]. Therefore, many efforts have been spent by the researchers in enhancing the stiffness and strength of PEEK by reinforcing with HA microparticles, HA whiskers and carbon fibers for load-bearing orthopedic applications [bib_ref] Tensile properties, tension-tension fatigue and biological response of polyetheretherketone-hydroxyapatite composites for load-bearing..., Bakar [/bib_ref] [bib_ref] Processing and tensile properties of hydroxyapatite-whisker-reinforced polyetheretherketone, Converse [/bib_ref] [bib_ref] The advantages of BioHPP polymer as superstructure material in oral implantology, Bechir [/bib_ref] [bib_ref] Investigation of mechanical impact behavior of short carbon-fiber-reinforced PEEK composites, Garcia-Gonzalez [/bib_ref] [bib_ref] First results of a new vacuum plasma sprayed (VPS) titanium-coated carbon/PEEK composite..., Hoppe [/bib_ref] [bib_ref] Suitability of carbon fiber-reinforced polyetheretherketone cages for use as anterior struts following..., Heary [/bib_ref] [bib_ref] Carbon fiber reinforced PEEK composites based on 3D-printing technology for orthopedic and..., Han [/bib_ref] [bib_ref] Carbon fibre/polyether ether ketone (CF/PEEK) implants in orthopaedic oncology, Laux [/bib_ref] [bib_ref] Use of carbon-fiber-reinforced composite implants in orthopedic surgery, Hak [/bib_ref] [bib_ref] Tension-tension fatigue behavior of hydroxyapatite reinforced polyetheretherketone composites, Tang [/bib_ref] [bib_ref] Mechanical properties and in vitro response of strontium-containing hydroxyapatite/polyetheretherketone composites, Wong [/bib_ref] [bib_ref] Does PEEK/HA enhance bone formation compared with PEEK in a sheep cervical..., Walsh [/bib_ref] [bib_ref] Formulation of a covalently bonded hydroxyapatite and poly (ether ether ketone) composite, Hughes [/bib_ref] [bib_ref] Evaluating the bioactivity of a hydroxyapatite-incorporated polyetheretherketone biocomposite, Ma [/bib_ref] [bib_ref] The influence of sterilization processes on the micromechanical properties of carbon fiber-reinforced..., Godora [/bib_ref] [bib_ref] Carbon fiber reinforced PEEK optima-A composite material biomechanical properties and wear/debris characteristics..., Steinberg [/bib_ref]. The elastic modulus of HA is 120.6 GPa [bib_ref] Ab initio elastic properties and tensile strength of crystalline hydroxyapatite, Ching [/bib_ref] , so it can be used as a reinforcement to stiffen PEEK. Abu Bakar et al. incorporated 5-40 vol% mHA into PEEK, resulting in microcomposites with improved stiffness [bib_ref] Tensile properties, tension-tension fatigue and biological response of polyetheretherketone-hydroxyapatite composites for load-bearing..., Bakar [/bib_ref] [bib_ref] Tension-tension fatigue behavior of hydroxyapatite reinforced polyetheretherketone composites, Tang [/bib_ref]. However, the tensile strength of PEEK-mHA microcomposites was smaller than that of pure PEEK. The tensile strength of PEEK decreased markedly from 78.30 MPa to 64.71 and 43.76 MPa by adding 10 vol% and 40 vol% mHA, respectively. The reduction in tensile strength of PEEK-mHA microcomposites was attributed to a poor mHA-PEEK interfacial bonding, leading to decohesion of mHA particles from the polymer matrix. Moreover, the inclusion of 40 vol% mHA into a ductile PEEK matrix led to brittle fracture [fig_ref] Figure 1: Multiple stages involved in the assessment of newly developed biomaterials for bone... [/fig_ref]. The final failure strain of PEEK reduced markedly from 28.93% to 19.23% and 0.98%, respectively by adding 10 vol% and 40 vol% mHA.
Very recently, Ma and Guo injection molded PEEK microcomposites filled with 10, 20, 30, and 40 wt% mHA. The composite samples were cultured with MC3T3-E1 cells, and then implanted into rabbit cranial defects [bib_ref] Evaluating the bioactivity of a hydroxyapatite-incorporated polyetheretherketone biocomposite, Ma [/bib_ref]. [fig_ref] Figure 1: Multiple stages involved in the assessment of newly developed biomaterials for bone... [/fig_ref] shows the tensile strength and modulus of elasticity of composites as a function of mHA content. This figure reveals an increase of stiffness but a decline in tensile strength of microcomposites with increasing mHA content. The PEEK/30 wt% mHA and PEEK/40 wt% mHA composites show higher elastic modulus than the low modulus range of cortical bone (7 GPa). However, the tensile strength of PEEK-40wt% mHA composite is smaller than the low tensile strength range of cortical bone, i.e., 50 MPa [bib_ref] Hydrostatically extruded HAPEX TM, Wang [/bib_ref]. So only PEEK/30 wt% mHA composite exhibits tensile properties close to cortical bone. This figure also reveals that that tensile strength of pure PEEK is 84 MPa, almost one-half of the upper limit of tensile strength of cortical bone (150 MPa) [bib_ref] Hydrostatically extruded HAPEX TM, Wang [/bib_ref]. This implies that pure PEEK can sustain the applied load. In the composite materials, polymeric matrix transfers the applied stress to the fillers for load-bearing capacity during tensile testing. Because of poor mHA-PEEK interfacial bonding, inefficient stress transfer between the matrix and mHA is expected. As a result, the PEEK matrix solely carries the load. However, the volume content of PEEK matrix decreases markedly by adding 30 wt% and 40 wt% mHA. This leads to a sharp decline in the tensile strength of PEEK/30 wt% mHA and PEEK/40 wt% mHA composites. As recognized, the interfacial bonding of ceramic fillers and the polymer matrix can be improved by adding coupling agents like silanes. However, silane agents are known to cause irritation of the eyes, skin and respiratory tract. Therefore, the use of coupling agents in biomedical implants must be avoided. [fig_ref] Figure 1: Multiple stages involved in the assessment of newly developed biomaterials for bone... [/fig_ref] ,b show the cell attachment and ALP activity of murine pre-osteoblastic cell line (MC3T3-E1) on pure PEEK, UHMWPE, and PEEK/30 wt% mHA composite at different time points. Apparently, the composite exhibits better cell adhesion and higher ALP activity level than pure PEEK and UHMWPE. [fig_ref] Figure 1: Multiple stages involved in the assessment of newly developed biomaterials for bone... [/fig_ref] -c show the respective histological examination of pure PEEK, UHMWPE, and PEEK/30 wt% mHA composite after 8 weeks of implantation. A layer of fibrous connective tissue can be readily seen in UHMWPE and PEEK [fig_ref] Figure 1: Multiple stages involved in the assessment of newly developed biomaterials for bone... [/fig_ref]. In contrast, new bone tissues are formed and contacted closely with the PEEK-30wt% mHA composite [fig_ref] Figure 1: Multiple stages involved in the assessment of newly developed biomaterials for bone... [/fig_ref]. Furthermore, the composite exhibits higher bone/implant contact ratio than UHMWPE and PEEK [fig_ref] Figure 1: Multiple stages involved in the assessment of newly developed biomaterials for bone... [/fig_ref]. Very recently, Ma and Guo injection molded PEEK microcomposites filled with 10, 20, 30, and 40 wt% mHA. The composite samples were cultured with MC3T3-E1 cells, and then implanted into rabbit cranial defects [bib_ref] Evaluating the bioactivity of a hydroxyapatite-incorporated polyetheretherketone biocomposite, Ma [/bib_ref]. [fig_ref] Figure 1: Multiple stages involved in the assessment of newly developed biomaterials for bone... [/fig_ref] shows the tensile strength and modulus of elasticity of composites as a function of mHA content. This figure reveals an increase of stiffness but a decline in tensile strength of microcomposites with increasing mHA content. The PEEK/30 wt% mHA and PEEK/40 wt% mHA composites show higher elastic modulus than the low modulus range of cortical bone (7 GPa). However, the tensile strength of PEEK-40wt% mHA composite is smaller than the low tensile strength range of cortical bone, i.e., 50 MPa [bib_ref] Hydrostatically extruded HAPEX TM, Wang [/bib_ref]. So only PEEK/30 wt% mHA composite exhibits tensile properties close to cortical bone. This figure also reveals that that tensile strength of pure PEEK is 84 MPa, almost one-half of the upper limit of tensile strength of cortical bone (150 MPa) [bib_ref] Hydrostatically extruded HAPEX TM, Wang [/bib_ref]. This implies that pure PEEK can sustain the applied load. In the composite materials, polymeric matrix transfers the applied stress to the fillers for load-bearing capacity during tensile testing. Because of poor mHA-PEEK interfacial bonding, inefficient stress transfer between the matrix and mHA is expected. As a result, the PEEK matrix solely carries the load. However, the volume content of PEEK matrix decreases markedly by adding 30 wt% and 40 wt% mHA. This leads to a sharp decline in the tensile strength of PEEK/30 wt% mHA and PEEK/40 wt% mHA composites. As recognized, the interfacial bonding of ceramic fillers and the polymer matrix can be improved by adding coupling agents like silanes. However, silane agents are known to cause irritation of the eyes, skin and respiratory tract. Therefore, the use of coupling agents in biomedical implants must be avoided. [fig_ref] Figure 1: Multiple stages involved in the assessment of newly developed biomaterials for bone... [/fig_ref] ,b show the cell attachment and ALP activity of murine pre-osteoblastic cell line (MC3T3-E1) on pure PEEK, UHMWPE, and PEEK/30 wt% mHA composite at different time points. Apparently, the composite exhibits better cell adhesion and higher ALP activity level than pure PEEK and UHMWPE. [fig_ref] Figure 1: Multiple stages involved in the assessment of newly developed biomaterials for bone... [/fig_ref] -c show the respective histological examination of pure PEEK, UHMWPE, and PEEK/30 wt% mHA composite after 8 weeks of implantation. A layer of fibrous connective tissue can be readily seen in UHMWPE and PEEK [fig_ref] Figure 1: Multiple stages involved in the assessment of newly developed biomaterials for bone... [/fig_ref]. In contrast, new bone tissues are formed and contacted closely with the PEEK-30wt% mHA composite [fig_ref] Figure 1: Multiple stages involved in the assessment of newly developed biomaterials for bone... [/fig_ref]. Furthermore, the composite exhibits higher bone/implant contact ratio than UHMWPE and PEEK [fig_ref] Figure 1: Multiple stages involved in the assessment of newly developed biomaterials for bone... [/fig_ref].
## Peek/carbon fiber microcomposites
PEEK-carbon fiber (CF) composites have been explored for orthopedic applications in posterior lumbar interbody fusion implants, screws, intramedullary nails, acetabular cups and dental implants [bib_ref] First results of a new vacuum plasma sprayed (VPS) titanium-coated carbon/PEEK composite..., Hoppe [/bib_ref] [bib_ref] Suitability of carbon fiber-reinforced polyetheretherketone cages for use as anterior struts following..., Heary [/bib_ref] [bib_ref] Carbon fiber reinforced PEEK composites based on 3D-printing technology for orthopedic and..., Han [/bib_ref] [bib_ref] Carbon fibre/polyether ether ketone (CF/PEEK) implants in orthopaedic oncology, Laux [/bib_ref] [bib_ref] Use of carbon-fiber-reinforced composite implants in orthopedic surgery, Hak [/bib_ref] [bib_ref] The influence of sterilization processes on the micromechanical properties of carbon fiber-reinforced..., Godora [/bib_ref] [bib_ref] Carbon fiber reinforced PEEK optima-A composite material biomechanical properties and wear/debris characteristics..., Steinberg [/bib_ref] [bib_ref] PEEK and CFR-PEEK as alternative bearing materials to UHMWPE in a fixed..., Brockett [/bib_ref] [bib_ref] Inflammatory response against different carbon fiber-reinforced PEEK wear particles compared with UHMWPE..., Utzschneider [/bib_ref] [bib_ref] Additively manufactured polyetheretherketone (PEEK) with carbon nanostructure reinforcement for biomedical structural applications, Alam [/bib_ref] [bib_ref] Stress shielding and fatigue limits of poly-ether-ether-ketone dental implants, Lee [/bib_ref] [bib_ref] Carbon-fiber-reinforced PEEK fixation system in the treatment of spine tumors: A preliminary..., Boriani [/bib_ref]. Pure PEEK is inadequate for those applications because of insufficient stiffness. In particular, PEEK/CF composites are useful for load-bearing applications such as femoral stems of hip joint prostheses and bone fracture plates [bib_ref] Proximal humeral fracture fixation: Multicenter study with carbon fiber peek plate, Rotini [/bib_ref] [bib_ref] Treatment of proximal humerus fractures with a CFR-PEEK plate: 2-year results of..., Schliemann [/bib_ref] [bib_ref] Compounds and composite materials, Green [/bib_ref] [bib_ref] Novel surface modifications of carbon fiber-reinforced polyetheretherketone hip stem in an ovine..., Nakahara [/bib_ref] [bib_ref] In vivo implant fixation of carbon fiber-reinforced PEEK hip prostheses in an..., Nakahara [/bib_ref]. PEEK/CF materials offer the possibility of altering the stiffness of implants to match the modulus of host bone by changing the volume fraction, length and orientation of reinforcing carbon fibers. As such, the elastic modulus of PEEK-CF composites can be tailored readily to reach the stiffness of cortical bone. The precursors generally used to manufacture commercial CFs include polyacrylonitrile (PAN) and pitch. PAN-based CFs can be classified into high strength fibers with a stiffness of 230-300 GPa and tensile strength of 3500-6400 MPa, and high modulus fibers with a stiffness of 370-590 GPa and tensile strength of 4000-5500 MPa. Pitch-based CFs prepared from petroleum products generally have larger diameter and lower strength than PAN-based CFs. The stiffness of pitch-based CFs varies from 50 to 900 GPa [bib_ref] Compounds and composite materials, Green [/bib_ref].
## Peek/carbon fiber microcomposites
PEEK-carbon fiber (CF) composites have been explored for orthopedic applications in posterior lumbar interbody fusion implants, screws, intramedullary nails, acetabular cups and dental implants [bib_ref] First results of a new vacuum plasma sprayed (VPS) titanium-coated carbon/PEEK composite..., Hoppe [/bib_ref] [bib_ref] Suitability of carbon fiber-reinforced polyetheretherketone cages for use as anterior struts following..., Heary [/bib_ref] [bib_ref] Carbon fiber reinforced PEEK composites based on 3D-printing technology for orthopedic and..., Han [/bib_ref] [bib_ref] Carbon fibre/polyether ether ketone (CF/PEEK) implants in orthopaedic oncology, Laux [/bib_ref] [bib_ref] Use of carbon-fiber-reinforced composite implants in orthopedic surgery, Hak [/bib_ref] [bib_ref] The influence of sterilization processes on the micromechanical properties of carbon fiber-reinforced..., Godora [/bib_ref] [bib_ref] Carbon fiber reinforced PEEK optima-A composite material biomechanical properties and wear/debris characteristics..., Steinberg [/bib_ref] [bib_ref] PEEK and CFR-PEEK as alternative bearing materials to UHMWPE in a fixed..., Brockett [/bib_ref] [bib_ref] Inflammatory response against different carbon fiber-reinforced PEEK wear particles compared with UHMWPE..., Utzschneider [/bib_ref] [bib_ref] Additively manufactured polyetheretherketone (PEEK) with carbon nanostructure reinforcement for biomedical structural applications, Alam [/bib_ref] [bib_ref] Stress shielding and fatigue limits of poly-ether-ether-ketone dental implants, Lee [/bib_ref] [bib_ref] Carbon-fiber-reinforced PEEK fixation system in the treatment of spine tumors: A preliminary..., Boriani [/bib_ref]. Pure PEEK is inadequate for those applications because of insufficient stiffness. In particular, PEEK/CF composites are useful for load-bearing applications such as femoral stems of hip joint prostheses and bone fracture plates [bib_ref] Proximal humeral fracture fixation: Multicenter study with carbon fiber peek plate, Rotini [/bib_ref] [bib_ref] Treatment of proximal humerus fractures with a CFR-PEEK plate: 2-year results of..., Schliemann [/bib_ref] [bib_ref] Compounds and composite materials, Green [/bib_ref] [bib_ref] Novel surface modifications of carbon fiber-reinforced polyetheretherketone hip stem in an ovine..., Nakahara [/bib_ref] [bib_ref] In vivo implant fixation of carbon fiber-reinforced PEEK hip prostheses in an..., Nakahara [/bib_ref]. PEEK/CF materials offer the possibility of altering the stiffness of implants to match the modulus of host bone by changing the volume fraction, length and orientation of reinforcing carbon fibers. As such, the elastic modulus of PEEK-CF composites can be tailored readily to reach the stiffness of cortical bone. The precursors generally used to manufacture commercial CFs include polyacrylonitrile (PAN) and pitch. PAN-based CFs can be classified into high strength fibers with a stiffness of 230-300 GPa and tensile strength of 3500-6400 MPa, and high modulus fibers with a stiffness of 370-590 GPa and tensile strength of 4000-5500 MPa. Pitch-based CFs prepared from petroleum products generally have larger diameter and lower strength than PAN-based CFs. The stiffness of pitch-based CFs varies from 50 to 900 GPa [bib_ref] Compounds and composite materials, Green [/bib_ref].
Endolign ® composites consist of PEEK matrix and continuous CFs (65 vol% [bib_ref] Compounds and composite materials, Green [/bib_ref]. Thus M.D. Endolign ® composite with a stiffness closer to cortical bone is more suitable for fabricating load-bearing implants. PEEK and preforms with high CF content and long fiber length cannot be processed in conventional injection molders to produce microcomposites. Continuous CFs reinforced PEEK composites are generally manufactured using compression molding, autoclave molding, filament winding, and pultrusion. Such high-temperature polymer composite processing techniques are typically developed for aerospace and automotive industries for forming components with complex geometries [bib_ref] Fiber-reinforced polymer composites: Manufacturing, properties, and applications, Rajak [/bib_ref].
The mechanical properties of PEEK composites reinforced with continuous CFs are anisotropic. Longitudinal tensile modulus and strength of unidirectional fiber-reinforced composite in which fibers align parallel to the loading direction are considerably higher than transverse tensile modulus and strength (fibers normal to the tensile loading direction). In contrast, the mechanical properties of PEEK composites reinforced with randomly oriented short carbon fibers (SCFs) are isotropic. SCFs are much cheaper than continuous CFs, and PEEK/SCF composite can be readily fabricated using conventional extrusion and injection molding techniques. The PEEK/30 wt% SCF composite exhibits a stiffness of 24 GPa and tensile strength of 214 MPa [bib_ref] Investigation of mechanical impact behavior of short carbon-fiber-reinforced PEEK composites, Garcia-Gonzalez [/bib_ref]. Very recently, Bonnheim et al. examined tensile and fatigue properties of orthopedic grade PEEK reinforced with 30 wt% PAN-and 30 wt% pitch-based SCFs. The stiffness of PAN-and pitch-based fibers was 540 GPa and 280 GPa, respectively [bib_ref] Effect of carbon fiber type on monotonic and fatigue properties of orthopedic..., Bonnheim [/bib_ref]. The elastic modulus, tensile strength, and fracture strain of injection molded PEEK/30 wt% SCF (PAN) are 18.5 ± 2.3 GPa, 192 ± 17 MPa and 1.9 ± 0.2%, while those of PEEK/30 wt%SCF (pitch) are 12.5 ± 1.3 GPa, 145 ± 9 MPa and 2.2 ± 0.2%, respectively. Rupp and coworkers employed FFF method to print PEEK/5 wt% SCF with a stiffness of 7.37 ± 1.22 GPa, thus reaching the low modulus range of cortical bone [bib_ref] Carbon fiber reinforced PEEK composites based on 3D-printing technology for orthopedic and..., Han [/bib_ref]. The close resemblance of the mechanical properties of PEEK/SCF composites with human cortical bone rendering them a potential biomaterial for orthopedic applications. [fig_ref] Table 1: Tensile properties of pure PEEK and its microcomposites [/fig_ref] summarizes the tensile properties of PEEK-based microcomposites.
## Peek/cf hip prostheses
In general, little work has been reported on the use of PEEK/CF composites for load-bearing hip stem applications. Furthermore, there have been concerns relating the inertness of PEEK/CF composites, which prevents the formation of bone tissues around the implants. Earlier study conducted by showed that the attachment and proliferation of primary human osteoblasts on PEEK/CF disks were not significantly different to those on Ti-6Al-4V alloy [bib_ref] Use of a novel carbon fibre composite material for the femoral stem..., Scotchford [/bib_ref]. However, the in vitro cell culture studies were conducted for short time periods, and the results could not directly reflect the safe use of composite implants in the clinical sector.
Nakahara et al. implanted HA-coated PEEK/65vol% CF (unidirectional) composite hip stem in an adult ovine model for 12 months. The HA-coated PEEK/CF hip stem exhibited bone on-growth fixation and minimal stress shielding. However, bone resorption and osteopenia were observed in HA-coated Ti-6Al-4V alloy hip stem [bib_ref] Novel surface modifications of carbon fiber-reinforced polyetheretherketone hip stem in an ovine..., Nakahara [/bib_ref]. In another study, they also conducted in vivo implant fixation of cementless and cemented PEEK/CF hip stems in an ovine model over 52 weeks of implantation. Uncemented HA-coated PEEK/CF stem showed bony ongrowth fixation. Cemented PEEK/CF stem without HA coating provided a stable long-term fixation showing no gaps at both the bone-cement and cement-stem interfaces [bib_ref] In vivo implant fixation of carbon fiber-reinforced PEEK hip prostheses in an..., Nakahara [/bib_ref]. Nakahara et al. also implanted HA-coated PEEK/CF into the femurs of rabbits. The interfacial shear strength of HA-coated PEEK/CF and uncoated PEEK/CF implants was determined using the pull-out test [bib_ref] Interfacial shear strength of bioactive-coated carbon fiber reinforced polyetheretherketone after in vivo..., Nakahara [/bib_ref]. After implantation for 6 weeks, the shear strength of HA-coated PEEK/CF implant (15.7 ± 4.5 MPa) was twice the strength of uncoated PEEK/CF implant (7.7 ± 1.8 MPa). At 12 weeks, the shear strength of HA-coated PEEK/CF and pristine PEEK/CF implants was found to be 17.4 ± 3.6 MPa and 8.3 ± 3.0 MPa, respectively. These results revealed the effectiveness of HA in enhancing the bone growth around the coated implant. Similarly, Suska et al. also indicated that plasma-sprayed HA-PEEK/CF implants inserted into the femurs and tibias of rabbits showing better osseointegration than uncoated PEEK/CF implants [bib_ref] Enhancement of CRF-PEEK osseointegration by plasma-sprayed hydroxyapatite: A rabbit model, Suska [/bib_ref]. In a recent study of Stubinger et al., HA-coated PEEK/CF implants inserted into the pelvis of sheep showed a significant improvement of osseointegration over uncoated PEEK/CF samples after 2 and 12 weeks of implantation [bib_ref] Titanium and hydroxyapatite coating of polyetheretherketone and carbon fiber-reinforced polyetheretherketone: A pilot..., Stubinger [/bib_ref].
UHMWPE has been widely used as a bearing surface material of hip prostheses articulating with a metallic or ceramic counterface. The wear debris released by UHMWPE acetabular cup into the surrounding tissue often leads to inflammation, causing osteolysis and implant failure [bib_ref] Particle disease: A current review of the biological mechanisms in periprosthetic osteolysis..., Sukur [/bib_ref]. PEEK exhibits a three times higher friction coefficient than UHMWPE [bib_ref] The influence of surface properties on sliding contact temperature and friction for..., Laux [/bib_ref] , rendering PEEK with poorer wear performance than UHMWPE. Taking advantages of high stiffness and strength, PEEK/SCF composite exhibits a higher wear resistance than pure PEEK [bib_ref] Influence of contact pressure, cross-shear and counterface material on the wear of..., Brockett [/bib_ref] [bib_ref] Wear studies on the likely performance of CFR-PEEK/CoCrMo for use as artificial..., Scholes [/bib_ref]. From the hip simulator testing for 10 million cycles, the wear rate of PEEK/30 wt% SCF (pitch) composite articulating against a zirconia ceramic head is almost two orders of magnitude lower than that of UHMWPE [bib_ref] Carbon fiber reinforced polyether ether ketone composite as a bearing surface for..., Wang [/bib_ref]. Thus PEEK/SCF composites can provide good stability for acetabular cup in hip prostheses sliding against hard femoral head. However, PEEK/30 wt% SCF (pitch) and PEEK/30 wt% SCF (PAN) composites exhibit higher wear rates than UHMWPE using a multidirectional cylinder-on-flat tribometer with bovine serum as lubricant. The multidirectional wear test simulates high contact stress conditions at the knee [bib_ref] Suitability and limitations of carbon fiber reinforced PEEK composites as bearing surfaces..., Wang [/bib_ref].
The wear resistance of polymeric materials is dependent on the applied stress, sliding distance, temperature, the presence of lubricant, etc. At a high applied stress or contact pressure, the wear rate increases markedly as expected. This is due to the removal of a large fraction of volume of material as wear debris. Recently, Brockett et al. studied the wear performance of PEEK and PEEK/30 wt% SCF (pitch) composite articulating with a clinically CoCrMo femoral bearing in a low conformity knee replacement using a six-station multidirectional pin-on-plate wear simulator. They demonstrated that PEEK and PEEK/30 wt% SCF composite exhibit very high wear rates under a low-conforming, high contact stress knee condition. So, they are unsuitable to replace UHMWPE in total knee replacement [bib_ref] PEEK and CFR-PEEK as alternative bearing materials to UHMWPE in a fixed..., Brockett [/bib_ref].
## Peek-based nanocomposites
## Peek-nha nanocomposites
As mentioned, human bones consists of 70% nHA platelets and 30% collagen fibrils. Thus, bionanocomposites for hard tissue regeneration are developed and designed by combining PEEK and nHA with nanometer dimension [bib_ref] Sintered hydroxyapatite/polyetheretherketone nanocomposites: Mechanical behavior and biocompatibility aspects, Li [/bib_ref] [bib_ref] Preparation of polyetheretherketone composites with nanohydroxyapatite rods and carbon nanofibers having high..., Chan [/bib_ref] [bib_ref] Polyetheretherketone hybrid composites with bioactive nanohydroxyapatite and multiwalled carbon nanotube fillers, Liu [/bib_ref] [bib_ref] Preparation, characterization, and in vitro osteoblast functions of a nano-hydroxyapatite/polyetheretherketone biocomposite as..., Ma [/bib_ref] [bib_ref] Characterization of polyetheretherketone-hydroxyapatite nanocomposite materials, Wang [/bib_ref] [bib_ref] Osseointegration of nanohydroxyapatite-or nano-calcium silicate-incorporated polyetheretherketone bioactive composites in vivo, Ma [/bib_ref] [bib_ref] Polyetheretherketone/ nano-fluorohydroxyapatite composite with antimicrobial activity and osseointegration properties, Wang [/bib_ref]. Nanohydroxyapatite can be prepared by means of co-precipitation, sol-gel, and hydrothermal techniques [bib_ref] Influence of hydrothermal synthesis parameters on the properties of hydroxyapatite nanoparticles, Kuśnieruk [/bib_ref]. It generally exhibits a rod-like feature [fig_ref] Figure 1: Multiple stages involved in the assessment of newly developed biomaterials for bone... [/fig_ref]. Synthetic nHA has been reported to promote the adhesion, proliferation and differentiation of osteoblasts, and new bone formation [bib_ref] Biomimetic and mesoporous nano-hydroxyapatite for bone tissue application: A short review, Morino [/bib_ref] [bib_ref] Optimizing nanohydroxyapatite nanocomposites for bone tissue engineering, Lowe [/bib_ref] [bib_ref] Biodegradable and biocompatible systems based on hydroxyapatite nanoparticles, Turon [/bib_ref] [bib_ref] Hydrothermal synthesis and biocompatibility study of highly crystalline carbonated hydroxyapatite nanorods, Xue [/bib_ref] [bib_ref] Hydrothermal synthesis and bio-mineralization of hydroxyapatite nanorods, Li [/bib_ref]. Furthermore, nHA favors more protein adsorption than mHA, leading to an initial cell attachment on its surface [bib_ref] A review of protein adsorption on bioceramics. Interf, Wang [/bib_ref]. Ma and coworkers mixed PEEK powders and nHA particles in a ball mill followed by injection molding [bib_ref] Preparation, characterization, and in vitro osteoblast functions of a nano-hydroxyapatite/polyetheretherketone biocomposite as..., Ma [/bib_ref] [bib_ref] Characterization of polyetheretherketone-hydroxyapatite nanocomposite materials, Wang [/bib_ref]. The stiffness of PEEK, PEEK/20 wt% nHA and PEEK/40wt% is reported to be 2.20 ± 0.17 GPa, 3.40 ± 0.20 GPa and 4.60 ± 0.12 GPa, respectively. The stiffness of ball-milled/injection molded PEEK is smaller than the modulus of PEEK reported in the literature, i.e., 3.8 GPa [bib_ref] High-performance nanocomposites based on polyetherketones, Díez-Pascual [/bib_ref]. The tensile strength of PEEK, PEEK/20 wt% nHA and PEEK/40 wt% is 84.0 ± 1.9 MPa, 81.0 ± 2.4 MPa and 75.0±2.7 MPa, respectively. Apparently, there exists no reinforcing effect of nHA for PEEK. This issue results from the mixing of PEEK powders and nHA for forming composites. Commercial PEEK powders are inadequate for injection molding purposes. On the contrary, Tjong and coworkers used injection molded grade PEEK pellets (PEEK-Optima, Invibio) as the matrix material of nanocomposites. The PEEK pellets and nHA were melt-mixed in a Brabender compounder. The extruded strands were chopped into pieces by a pelletizer and loaded into Brabender again to achieve homogeneous filler dispersion in the polymer matrix. The compounded pellets were finally injection molded to produce composite samples. The PEEK/nHA nanocomposites showed a slight improvement in tensile strength by adding 10 wt% nHA and 20 wt% nHA at the expense of tensile ductility. Thereafter, it decreased with increasing nHA content, i.e., ≥ 30 wt% nHA [fig_ref] Figure 1: Multiple stages involved in the assessment of newly developed biomaterials for bone... [/fig_ref] [bib_ref] Preparation of polyetheretherketone composites with nanohydroxyapatite rods and carbon nanofibers having high..., Chan [/bib_ref]. Ma and coworkers mixed PEEK powders and nHA particles in a ball mill followed by injection molding [bib_ref] Preparation, characterization, and in vitro osteoblast functions of a nano-hydroxyapatite/polyetheretherketone biocomposite as..., Ma [/bib_ref] [bib_ref] Characterization of polyetheretherketone-hydroxyapatite nanocomposite materials, Wang [/bib_ref]. The stiffness of PEEK, PEEK/20 wt% nHA and PEEK/40wt% is reported to be 2.20 ± 0.17 GPa, 3.40 ± 0.20 GPa and 4.60 ± 0.12 GPa, respectively. The stiffness of ball-milled/injection molded PEEK is smaller than the modulus of PEEK reported in the literature, i.e., 3.8 GPa [bib_ref] High-performance nanocomposites based on polyetherketones, Díez-Pascual [/bib_ref]. The tensile strength of PEEK, PEEK/20 wt% nHA and PEEK/40 wt% is 84.0 ± 1.9 MPa, 81.0 ± 2.4 MPa and 75.0±2.7 MPa, respectively. Apparently, there exists no reinforcing effect of nHA for PEEK. This issue results from the mixing of PEEK powders and nHA for forming composites. Commercial PEEK powders are inadequate for injection molding purposes. On the contrary, Tjong and coworkers used injection molded grade PEEK pellets (PEEK-Optima, Invibio) as the matrix material of nanocomposites. The PEEK pellets and nHA were melt-mixed in a Brabender compounder. The extruded strands were chopped into pieces by a pelletizer and loaded into Brabender again to achieve homogeneous filler dispersion in the polymer matrix. The compounded pellets were finally injection molded to produce composite samples. The PEEK/nHA nanocomposites showed a slight improvement in tensile strength by adding 10 wt% nHA and 20 wt% nHA at the expense of tensile ductility. Thereafter, it decreased with increasing nHA content, i.e., ≥ 30 wt% nHA [fig_ref] Figure 1: Multiple stages involved in the assessment of newly developed biomaterials for bone... [/fig_ref] [bib_ref] Preparation of polyetheretherketone composites with nanohydroxyapatite rods and carbon nanofibers having high..., Chan [/bib_ref].
The elastic modulus of PEEK is 3.87 ± 0.10 GPa and increases to 4.34 ± 0.08 GPa and 4.92 ± 0.06 GPa by adding 10 wt% and 20 wt% nHA, respectively. The stiffness of PEEK/40 wt% nHA reaches 7.85 ± 0.11GPa, being higher than the lower modulus range of cortical bone. However, PEEK/40wt% nHA with a tensile ductility of 0.69 ± 0.21% is very brittle; this value is smaller than the fracture strain of cortical bone ranging from 1-3%. So, this stiff and brittle composite is unsuitable for bone replacement. The stiffness of PEEK/30wt% nHA is 6.20 ± 0.13 GPa, while the tensile strength (70.56 ± 3.22 MPa) and fracture ductility (2.71%) of this nanocomposite meet the tensile properties of cortical bone. The elastic modulus of PEEK/30 wt% nHA can be further improved to 6.54 GPa by adding 2wt% carbon nanofibers (CNFs). The incorporation of 30 wt% nHA into PEEK promotes the adhesion of human osteoblastic cells (Saos-2) greatly [fig_ref] Figure 1: Multiple stages involved in the assessment of newly developed biomaterials for bone... [/fig_ref]. Comparing with pure PEEK, bone cell viability is dramatically improves by adding 20 wt% and 30 wt% nHA to PEEK as revealed by 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-1) assay [fig_ref] Figure 1: Multiple stages involved in the assessment of newly developed biomaterials for bone... [/fig_ref]. results from the mixing of PEEK powders and nHA for forming composites. Commercial PEEK powders are inadequate for injection molding purposes. On the contrary, Tjong and coworkers used injection molded grade PEEK pellets (PEEK-Optima, Invibio) as the matrix material of nanocomposites. The PEEK pellets and nHA were melt-mixed in a Brabender compounder. The extruded strands were chopped into pieces by a pelletizer and loaded into Brabender again to achieve homogeneous filler dispersion in the polymer matrix. The compounded pellets were finally injection molded to produce composite samples. The PEEK/nHA nanocomposites showed a slight improvement in tensile strength by adding 10 wt% nHA and 20 wt% nHA at the expense of tensile ductility. Thereafter, it decreased with increasing nHA content, i.e., ≥ 30 wt% nHA [fig_ref] Figure 1: Multiple stages involved in the assessment of newly developed biomaterials for bone... [/fig_ref] [bib_ref] Preparation of polyetheretherketone composites with nanohydroxyapatite rods and carbon nanofibers having high..., Chan [/bib_ref]. The elastic modulus of PEEK is 3.87 ± 0.10 GPa and increases to 4.34 ± 0.08 GPa and 4.92 ± 0.06 GPa by adding 10 wt% and 20 wt% nHA, respectively. The stiffness of PEEK/40 wt% nHA reaches 7.85 ± 0.11GPa, being higher than the lower modulus range of cortical bone. However, PEEK/40wt% nHA with a tensile ductility of 0.69 ± 0.21% is very brittle; this value is smaller than the fracture strain of cortical bone ranging from 1-3%. So, this stiff and brittle composite is unsuitable for bone replacement. The stiffness of PEEK/30wt% nHA is 6.20 ± 0.13 GPa, while the tensile strength (70.56 ± 3.22 MPa) and fracture ductility (2.71%) of this nanocomposite meet the tensile properties of cortical Ma et al. studied in vitro cell adhesion, proliferation and differentiation of murine MC3T3-E1 pre-osteoblasts on PEEK/40 wt% nHA despite its poor stiffness (4.60 ± 0.12 GPa), i.e., smaller than the low modulus range of cortical bone [bib_ref] Preparation, characterization, and in vitro osteoblast functions of a nano-hydroxyapatite/polyetheretherketone biocomposite as..., Ma [/bib_ref]. Comparing with pure PEEK and UHMWPE, PEEK/40 wt% nHA nanocomposite favors the adhesion and proliferation of MC3T3-E1 cells. [fig_ref] Figure 1: Multiple stages involved in the assessment of newly developed biomaterials for bone... [/fig_ref] -d show the real time polymerase chain reaction (RT-PCR) analysis of osteogenic marker genes of MC3T3-E1 cells cultured on PEEK, UHMWPE and PEEK/40 wt% nHA samples for different time points. PEEK/40 wt% nHA induces higher expression levels of osteogenic differentiation-related genes. In general, the differentiation of pre-osteoblasts occurs via three phases, i.e., proliferation, maturation of matrix, and mineralization [bib_ref] The characteristics of human bone-derived cells (HBDCS) during osteogenesis in vitro, Wrobel [/bib_ref]. During the proliferation phase, osteoblasts secrete bone matrix proteins including collagen type 1 (COL1), osteopontin (OPN) and fibronectin. Subsequently, they mature the extracellular matrix (ECM) with ALP and collagen. The final phase involves the mineralization of bone by expressing osteocalcin (OC), facilitating calcium phosphate deposition [bib_ref] Osteoblast differentiation and bone matrix formation in vivo and in vitro, Blair [/bib_ref]. From [fig_ref] Figure 1: Multiple stages involved in the assessment of newly developed biomaterials for bone... [/fig_ref] , a higher expression of ALP is observed on PEEK/40 wt% nHA compared to PEEK and UHMWPE at 7 and 14 days. The ALP activity of all samples then decreases at day 21 as expected, because ALP is an early marker of differentiation of pre-osteoblasts. Furthermore, bioactive nHA fillers are effective to promote the differentiation of osteoblasts. As such, PEEK/40 wt% nHA nanocomposite shows a higher COL1 and OPN expressions than pure PEEK and UHMWPE [fig_ref] Figure 1: Multiple stages involved in the assessment of newly developed biomaterials for bone... [/fig_ref]. Finally, PEEK/40 wt% nHA nanocomposite exhibits the highest OC expression level at day 21 associated with the mineralization of matrix [fig_ref] Figure 1: Multiple stages involved in the assessment of newly developed biomaterials for bone... [/fig_ref]. Recently, Zhao et al. also reported that melt-mixed/injection molded PEEK/40 wt% nHA composite exhibits enhanced adhesion and proliferation of human Ma et al. studied in vitro cell adhesion, proliferation and differentiation of murine MC3T3-E1 pre-osteoblasts on PEEK/40 wt% nHA despite its poor stiffness (4.60 ± 0.12 GPa), i.e., smaller than the low modulus range of cortical bone [bib_ref] Preparation, characterization, and in vitro osteoblast functions of a nano-hydroxyapatite/polyetheretherketone biocomposite as..., Ma [/bib_ref]. Comparing with pure PEEK and UHMWPE, PEEK/40 wt% nHA nanocomposite favors the adhesion and proliferation of MC3T3-E1 cells. [fig_ref] Figure 1: Multiple stages involved in the assessment of newly developed biomaterials for bone... [/fig_ref] -d show the real time polymerase chain reaction (RT-PCR) analysis of osteogenic marker genes of MC3T3-E1 cells cultured on PEEK, UHMWPE and PEEK/40 wt% nHA samples for different time points. PEEK/40 wt% nHA induces higher expression levels of osteogenic differentiation-related genes. In general, the differentiation of pre-osteoblasts occurs via three phases, i.e., proliferation, maturation of matrix, and mineralization [bib_ref] The characteristics of human bone-derived cells (HBDCS) during osteogenesis in vitro, Wrobel [/bib_ref]. During the proliferation phase, osteoblasts secrete bone matrix proteins including collagen type 1 (COL1), osteopontin (OPN) and fibronectin. Subsequently, they mature the extracellular matrix (ECM) with ALP and collagen. The final phase involves the mineralization of bone by expressing osteocalcin (OC), facilitating calcium phosphate deposition [bib_ref] Osteoblast differentiation and bone matrix formation in vivo and in vitro, Blair [/bib_ref]. From [fig_ref] Figure 1: Multiple stages involved in the assessment of newly developed biomaterials for bone... [/fig_ref] , a higher expression of ALP is observed on PEEK/40 wt% nHA compared to PEEK and UHMWPE at 7 and 14 days. The ALP activity of all samples then decreases at day 21 as expected, because ALP is an early marker of differentiation of pre-osteoblasts. Furthermore, bioactive nHA fillers are effective to promote the differentiation of osteoblasts. As such, PEEK/40 wt% nHA nanocomposite shows a higher COL1 and OPN expressions than pure PEEK and UHMWPE [fig_ref] Figure 1: Multiple stages involved in the assessment of newly developed biomaterials for bone... [/fig_ref]. Finally, PEEK/40 wt% nHA nanocomposite exhibits the highest OC expression level at day 21 associated with the mineralization of matrix [fig_ref] Figure 1: Multiple stages involved in the assessment of newly developed biomaterials for bone... [/fig_ref]. Recently, Zhao et al. also reported that melt-mixed/injection molded PEEK/40 wt% nHA composite exhibits enhanced adhesion and proliferation of human osteoblast-like MG-63 cells than pure PEEK [bib_ref] Response of human osteoblast to n-HA/PEEK-Quantitative proteomic study of bio-effects of nano-hydroxyapatite..., Zhao [/bib_ref]. Ma et al. also implanted PEEK and PEEK/40wt% nHA into full-thickness cranial defects of rabbits for 4 and 8 weeks. For comparison, injection molded PEEK/40 wt% calcium silicate (n-CS) was also implanted into the defect model [bib_ref] Osseointegration of nanohydroxyapatite-or nano-calcium silicate-incorporated polyetheretherketone bioactive composites in vivo, Ma [/bib_ref]. [fig_ref] Figure 1: Multiple stages involved in the assessment of newly developed biomaterials for bone... [/fig_ref] shows histological micrographs of PEEK and PEEK/40 wt% nHA samples after 4 and 8 weeks of implantation. There exists no direct bone contact for pure PEEK at 4 and 8 weeks. However, a layer of fibrous connective tissue as indicated by the black arrows can be readily seen. The fibrous tissue are commonly observed when foreign materials are implanted into a mammalian body. In contrast, bone tissues are formed on the PEEK/40 wt% nHA composite and contact directly with the implant surface as indicated by white arrows. The direct contact between the bone and implant, and the lack of fibrous connective tissue, demonstrating good osseointegration of the implant. [fig_ref] Figure 1: Multiple stages involved in the assessment of newly developed biomaterials for bone... [/fig_ref] reveals that both the PEEK/40 wt% nHA and PEEK/40 wt% n-CS implants exhibit higher bone contact than pure PEEK, especially for PEEK/40wt% n-CS. Ma et al. also implanted PEEK and PEEK/40wt% nHA into full-thickness cranial defects of rabbits for 4 and 8 weeks. For comparison, injection molded PEEK/40 wt% calcium silicate (n-CS) was also implanted into the defect model [bib_ref] Osseointegration of nanohydroxyapatite-or nano-calcium silicate-incorporated polyetheretherketone bioactive composites in vivo, Ma [/bib_ref]. [fig_ref] Figure 1: Multiple stages involved in the assessment of newly developed biomaterials for bone... [/fig_ref] shows histological micrographs of PEEK and PEEK/40 wt% nHA samples after 4 and 8 weeks of implantation. There exists no direct bone contact for pure PEEK at 4 and 8 weeks. However, a layer of fibrous connective tissue as indicated by the black arrows can be readily seen. The fibrous tissue are commonly observed when foreign materials are implanted into a mammalian body. In contrast, bone tissues are formed on the PEEK/40 wt% nHA composite and contact directly with the implant surface as indicated by white arrows. The direct contact between the bone and implant, and the lack of fibrous connective tissue, demonstrating good osseointegration of the implant. [fig_ref] Figure 1: Multiple stages involved in the assessment of newly developed biomaterials for bone... [/fig_ref] reveals that both the PEEK/40 wt% nHA and PEEK/40 wt% n-CS implants exhibit higher bone contact than pure PEEK, especially for PEEK/40wt% n-CS.
for pure PEEK at 4 and 8 weeks. However, a layer of fibrous connective tissue as indicated by the black arrows can be readily seen. The fibrous tissue are commonly observed when foreign materials are implanted into a mammalian body. In contrast, bone tissues are formed on the PEEK/40 wt% nHA composite and contact directly with the implant surface as indicated by white arrows. The direct contact between the bone and implant, and the lack of fibrous connective tissue, demonstrating good osseointegration of the implant. [fig_ref] Figure 1: Multiple stages involved in the assessment of newly developed biomaterials for bone... [/fig_ref] reveals that both the PEEK/40 wt% nHA and PEEK/40 wt% n-CS implants exhibit higher bone contact than pure PEEK, especially for PEEK/40wt% n-CS. Apart from osseointegration, the resistance to bacterial infection is another factor influencing the clinical success of implants. In particular, the failure of dental implants is associated with bacterial colonization and biofilm formation, leading to the inflammation and deterioration of the tissue implant integration [bib_ref] In vitro Investigation of the effect of oral bacteria in the surface..., Sridhar [/bib_ref]. Streptococcus mutans is a bacterial species residing in the human oral cavity, forming biofilm on dental implants and causing peri-implant diseases [bib_ref] Dental implants with anti-biofilm properties: A pilot study for developing a new..., Ghensi [/bib_ref]. Recently, Wang et al. fabricated PEEK composite filled with 40 wt% nano-fluorohydroxyapatite (nano-FHA) using compression molding process. Fluorine was incorporated into nHA to prevent bacterial colonization [bib_ref] Polyetheretherketone/ nano-fluorohydroxyapatite composite with antimicrobial activity and osseointegration properties, Wang [/bib_ref]. Some PEEK and PEEK/nano-FHA samples were blasted with TiO2 particles for surface roughening. The PEEK/nano-FHA composite promoted the adhesion, proliferation and differentiation of human MG63 osteoblasts as expected. Moreover, hMSCs were also seeded on these samples. The hMSC cells seeded on rough PEEK and PEEK/nano-FHA surfaces showed more brighter red staining than smooth sample groups at 21 days as revealed by Alizarin Red S staining, demonstrating mineralization of hMSCs at the final differentiation stage [fig_ref] Figure 1: Multiple stages involved in the assessment of newly developed biomaterials for bone... [/fig_ref]. Alizarin red agent stained the cells containing calcium deposits in dark red color. [fig_ref] Figure 2: The design and fabrication of polyetheretherketone patient-specific implants using CAD/CAM system for... [/fig_ref] ,b showed the growth of S. mutans and biofilm formation on rough PEEK and PEEK/nano-FHA as determined by bacteria live/dead assay. Dead bacteria were fluoresced red and living cells with intact cell membranes were labeled green. The results were analyzed with spectrophotometry and confocal laser scanning microscopy (CLSM). In [fig_ref] Figure 2: The design and fabrication of polyetheretherketone patient-specific implants using CAD/CAM system for... [/fig_ref] , few bacteria were attached on pure PEEK and composite samples Apart from osseointegration, the resistance to bacterial infection is another factor influencing the clinical success of implants. In particular, the failure of dental implants is associated with bacterial colonization and biofilm formation, leading to the inflammation and deterioration of the tissue implant integration [bib_ref] In vitro Investigation of the effect of oral bacteria in the surface..., Sridhar [/bib_ref]. Streptococcus mutans is a bacterial species residing in the human oral cavity, forming biofilm on dental implants and causing peri-implant diseases [bib_ref] Dental implants with anti-biofilm properties: A pilot study for developing a new..., Ghensi [/bib_ref]. Recently, Wang et al. fabricated PEEK composite filled with 40 wt% nano-fluorohydroxyapatite (nano-FHA) using compression molding process. Fluorine was incorporated into nHA to prevent bacterial colonization [bib_ref] Polyetheretherketone/ nano-fluorohydroxyapatite composite with antimicrobial activity and osseointegration properties, Wang [/bib_ref]. Some PEEK and PEEK/nano-FHA samples were blasted with TiO 2 particles for surface roughening. The PEEK/nano-FHA composite promoted the adhesion, proliferation and differentiation of human MG63 osteoblasts as expected. Moreover, hMSCs were also seeded on these samples. The hMSC cells seeded on rough PEEK and PEEK/nano-FHA surfaces showed more brighter red staining than smooth sample groups at 21 days as revealed by Alizarin Red S staining, demonstrating mineralization of hMSCs at the final differentiation stage [fig_ref] Figure 1: Multiple stages involved in the assessment of newly developed biomaterials for bone... [/fig_ref]. Alizarin red agent stained the cells containing calcium deposits in dark red color. [fig_ref] Figure 2: The design and fabrication of polyetheretherketone patient-specific implants using CAD/CAM system for... [/fig_ref] ,b showed the growth of S. mutans and biofilm formation on rough PEEK and PEEK/nano-FHA as determined by bacteria live/dead assay. Dead bacteria were fluoresced red and living cells with intact cell membranes were labeled green. The results were analyzed with spectrophotometry and confocal laser scanning microscopy (CLSM). In [fig_ref] Figure 2: The design and fabrication of polyetheretherketone patient-specific implants using CAD/CAM system for... [/fig_ref] , few bacteria were attached on pure PEEK and composite samples at 1h. However, bacterial colonies on PEEK increased sharply with time up 24 h, while low bacterial densities developed on the PEEK/nano-FHA, so forming a surface resistant to bacterial adhesion. The CLSM image of PEEK revealed the formation of biofilm with intense green fluorescence on its surface at prolonged culture period of 14 days. In contrast, numerous dead bacterial cells with red fluorescence were seen on the PEEK/nano-FHA surface [fig_ref] Figure 2: The design and fabrication of polyetheretherketone patient-specific implants using CAD/CAM system for... [/fig_ref]. From in vivo beagle dog model, rough PEEK/nano-FHA implant showed good bioactivity, osseointegration, and bone-implant contact.
## Peek-metal oxide nanocomposites
Metal oxides such as titania and zinc oxide (ZnO) are wide-bandgap semiconductors having photocatalytic properties. Excitation those oxides with photons of energy greater than the band-gap, [fig_ref] Figure 1: Multiple stages involved in the assessment of newly developed biomaterials for bone... [/fig_ref]. (a) The formation of mineralized extracellular matrix of hMSCs cultivated on different PEEK-based samples after staining with Alizarin Red S at day 21. Alizarin red dye stained the cells containing calcium deposits in dark red color. (b) Mineralization of hMSCs seeded on both smooth and rough PEEK as well as its composite groups at day 21 using Alizarin Red S assay. ** represents p < 0.01, and # represents p < 0.05 compared with other groups. Reproduced from [bib_ref] Polyetheretherketone/ nano-fluorohydroxyapatite composite with antimicrobial activity and osseointegration properties, Wang [/bib_ref] with permission of Elsevier.
## Peek-metal oxide nanocomposites
Metal oxides such as titania and zinc oxide (ZnO) are wide-bandgap semiconductors having photocatalytic properties. Excitation those oxides with photons of energy greater than the band-gap, an electron in the valence band (VB) is ejected and jumped to the conduction band, producing a positive hole in the VB. As the photogenerated electron-hole pairs arrive the semiconductor surface,
## Peek-metal oxide nanocomposites
Metal oxides such as titania and zinc oxide (ZnO) are wide-bandgap semiconductors having photocatalytic properties. Excitation those oxides with photons of energy greater than the band-gap, an electron in the valence band (VB) is ejected and jumped to the conduction band, producing a positive hole in the VB. As the photogenerated electron-hole pairs arrive the semiconductor surface, they react with surface-adsorbed water and oxygen to generate reactive oxygen species (ROS) like hydroxyl (- OH), superoxide anion (O 2− ), and hydrogen peroxide (H 2 O 2 ). [bib_ref] Generation and detection of reactive oxygen species in photocatalysis, Nosaka [/bib_ref]. Photogenerated ROS can inactivate microbial pathogens, rendering TiO 2 and ZnO effective nanomaterials for bacterial killing [bib_ref] Antimicrobial activity of zinc and titanium dioxide nanoparticles against biofilm-producing methicillin-resistant Staphylococcus..., Jesline [/bib_ref] [bib_ref] Comparison of infectious agents susceptibility to photocatalytic effects of nanosized titanium and..., Bogdan [/bib_ref]. Because of the wide-bandgap, ROS can be induced on TiO 2 under ultraviolet (UV) light irradiation. This implies that visible light with long wavelengths is incapable of generating ROS on TiO 2 for bacterial inactivation. UV radiation is known to be harmful to humans, especially for the skin and eyes. On the contrary, ZnO can generate ROS under visible light and even in the dark due to the presence of surface vacancy defects [bib_ref] Insight into the mechanism of antibacterial activity of ZnO: Surface defects mediated..., Prasanna [/bib_ref]. Moreover, ZnO can release Zn 2+ ions upon direct contact with bacteria strains, thereby damaging their cell membranes [bib_ref] Review on zinc oxide nanoparticles: Antibacterial activity and toxicity mechanism, Sirelkhatim [/bib_ref]. Thus, ZnO nanoparticles are potential fillers for fabricating antibacterial PEEK-based nanocomposites for orthopedic applications. Surgical site infections due to bacterial contamination of medical implants can lead to a life-threatening postoperative complication [bib_ref] Implantable device related infection, Vanepps [/bib_ref]. The bacterial infections have motivated the researchers to develop medical implants with bactericidal activity.
Díez-Pascual and Díez-Vicente fabricated PEEK/ZnO and silane-modified PEEK/ZnO nanocomposites using melt-mixing process.
To improve interfacial filler-matrix bonding, ZnO nanoparticles (NPs) were surface modified with 5,6-epoxyhexyltriethoxysilane (EHTES), methanol and hydrochloric acid to yield ZnO−CH 2 OH. Meanwhile, PEEK was treated with sodium borohydride (NaBH 4 ) and dimethyl sulfoxide to form PEEK−COOH [bib_ref] Development of nanocomposites reinforced with carboxylated poly (ether ether ketone) grafted to..., Díez-Pascual [/bib_ref]. The PEEK−COOH was grafted to the surface of ZnO−CH 2 OH to create PEEK−CO−O−CH 2 −ZnO, terming as PCOZnO. The PCOZnO of desired contents were melt-mixed with PEEK to form modified PEEK nanocomposites. EHTES is toxic to humans by irritating the skin, eyes and respiratory tract system. NaBH 4 is explosive and toxic to human cells. The use of those chemical regents should be restricted for fabricating human implants. [fig_ref] Figure 1: Multiple stages involved in the assessment of newly developed biomaterials for bone... [/fig_ref] -d show the tensile properties of PEEK/ZnO and modified PEEK/ZnO nanocomposites. The stiffness and tensile strength of PEEK/ZnO nanocomposites improve slightly by adding 1.0 and 2.5 wt% ZnO NPs. At 5.0 wt% ZnO, the composite exhibits a decline in both the tensile modulus and strength due to the agglomeration of ZnO NPs. However, elastic modulus and tensile strength of modified PEEK/ZnO nanocomposites increase with increasing filler content at the expense of tensile elongation and toughness. The improvement in tensile strength and stiffness are attributed to enhanced interfacial filler-matrix bonding. [fig_ref] Figure 2: The design and fabrication of polyetheretherketone patient-specific implants using CAD/CAM system for... [/fig_ref] shows the antibacterial activity of PEEK/ZnO and modified PEEK/ZnO nanocomposites against Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli. As recognized, S. aureus often induces implant infections, which are hard to treat due to its antibiotic resistance and ability to form biofilm [bib_ref] Implant infections: Adhesion, biofilm formation and immune evasion, Arciola [/bib_ref]. Pure PEEK exhibits no bactericidal activity as expected. The survival ratio of both bacterial strains decreases with increasing nanofiller content, especially at filler contents ≥ 2.5 wt%. PEEK/ZnO and modified PEEK/ZnO nanocomposites exhibit higher antibacterial efficacy against E. coli than S. aureus. This is due to a difference in the cell wall structures of both bacterial strains. The antibacterial efficacy of modified PEEK/ZnO nanocomposites is stronger than PEEK/ZnO counterparts due to their improved filler dispersion. In another study, Díez-Pascual et al. modified ZnO NPs with 3-anilinopropyltrimethoxysilane (APTMS). The silane modified ZnO NPs (mZnO) were mixed with pure PEEK followed by compressing molding. Similarly, the bactericidal activity against S. aureus and E. coli was enhanced with increasing ZnO or mZnO content [bib_ref] Development and characterization of novel poly (ether ether ketone)/ ZnO bionanocomposites, Díez-Pascual [/bib_ref].
## Peek-cnt nanocomposites
Carbonaceous nanofilers like graphene and CNTs exhibit extraordinarily high elastic modulus and tensile strength as well as large aspect ratio. Graphene and CNTs generally have an elastic modulus of about 1 TPa [bib_ref] Tensile loading of ropes of single wall carbon nanotubes and their mechanical..., Yu [/bib_ref] [bib_ref] Measurement of the elastic properties and intrinsic strength of monolayer graphene, Lee [/bib_ref] , so they can be used to reinforce polymers at low filler loadings [bib_ref] Carbon-based polymer nanocomposites for high-performance applications, Díez-Pascual [/bib_ref] [bib_ref] Multifunctional polymer nanocomposites reinforced by aligned carbon nanomaterials, Wu [/bib_ref]. Single walled CNT (SWNT) is formed by wrapping a graphene sheet into a cylinder with a diameter of 1-2 nm. Multi walled CNT (MWNT) is generated by rolling multiple graphene sheets into concentric cylinders. SWNTs usually agglomerate into ropes or bundles due to van der Waals forces between carbon atoms. As such, SWNTs are difficult to disperse uniformly as independent nanotubes in the matrix of polymer nanocomposites. In contrast, MWNTs have weaker van der Waals forces, showing less tendency to form ropes and bundles. MWNTs exhibit a very high elastic modulus
## Peek-cnt nanocomposites
Carbonaceous nanofilers like graphene and CNTs exhibit extraordinarily high elastic modulus and tensile strength as well as large aspect ratio. Graphene and CNTs generally have an elastic modulus of about 1 TPa [bib_ref] Tensile loading of ropes of single wall carbon nanotubes and their mechanical..., Yu [/bib_ref] [bib_ref] Measurement of the elastic properties and intrinsic strength of monolayer graphene, Lee [/bib_ref] , so they can be used to reinforce polymers at low filler loadings [bib_ref] Carbon-based polymer nanocomposites for high-performance applications, Díez-Pascual [/bib_ref] [bib_ref] Multifunctional polymer nanocomposites reinforced by aligned carbon nanomaterials, Wu [/bib_ref]. Single walled CNT (SWNT) is formed by wrapping a graphene sheet into a cylinder with a diameter of 1-2 nm. Multi walled CNT (MWNT) is generated by rolling multiple graphene sheets into concentric cylinders. SWNTs usually agglomerate into ropes or bundles due to van der Waals forces between carbon atoms. As such, SWNTs are difficult to disperse uniformly as independent nanotubes in the matrix of polymer nanocomposites. In contrast, MWNTs have weaker van der Waals forces, showing less tendency to form ropes and bundles. MWNTs exhibit a very high elastic modulus
## Peek-cnt nanocomposites
Carbonaceous nanofilers like graphene and CNTs exhibit extraordinarily high elastic modulus and tensile strength as well as large aspect ratio. Graphene and CNTs generally have an elastic modulus of about 1 TPa [bib_ref] Tensile loading of ropes of single wall carbon nanotubes and their mechanical..., Yu [/bib_ref] [bib_ref] Measurement of the elastic properties and intrinsic strength of monolayer graphene, Lee [/bib_ref] , so they can be used to reinforce polymers at low filler loadings [bib_ref] Carbon-based polymer nanocomposites for high-performance applications, Díez-Pascual [/bib_ref] [bib_ref] Multifunctional polymer nanocomposites reinforced by aligned carbon nanomaterials, Wu [/bib_ref]. Single walled CNT (SWNT) is formed by wrapping a graphene sheet into a cylinder with a diameter of 1-2 nm. Multi walled CNT (MWNT) is generated by rolling multiple graphene sheets into concentric cylinders. SWNTs usually agglomerate into ropes or bundles due to van der Waals forces between carbon atoms. As such, SWNTs are difficult to disperse uniformly as independent nanotubes in the matrix of polymer nanocomposites. In contrast, MWNTs have weaker van der Waals forces, showing less tendency to form ropes and bundles. MWNTs exhibit a very high elastic modulus of 0.9 TPa and tensile strength of 150 GPa [bib_ref] Direct mechanical measurement of the tensile strength and elastic modulus of multiwalled..., Demczyk [/bib_ref]. Thus, MWNTs are usually used to reinforce polymers for industrial and biomedical applications. Vapor grown carbon nanofiber (VGCNF) comprises of different arrangement of graphitic layers in herringbone and tubular features, with diameters ranging from 50 to 500 nm [bib_ref] Comparative study of herringbone and stacked-cup carbon nanofibers, Kim [/bib_ref] [bib_ref] Influence of structural and textural parameters of carbon nanofibers on their capacitive..., Moyseowicz [/bib_ref]. The mechanical properties of CNFs are inferior to those of MWNTs due to the presence of more crystalline defects. From biomedical perspective, carbonaceous nanomaterials promote the adhesion, growth and differentiation of bone cells [bib_ref] Applications of carbon nanotubes in bone regenerative medicine, Tanaka [/bib_ref] [bib_ref] Recent advances in the use of carbon nanotubes as smart biomaterials, Menezes [/bib_ref] [bib_ref] Cell attachment and spreading on carbon nanotubes is facilitated by integrin binding, Imaninezhad [/bib_ref] [bib_ref] Carbon nanostructures in bone tissue engineering, Perkins [/bib_ref] [bib_ref] Graphene-induced osteogenic differentiation is mediated by the integrin/FAK axis, Xie [/bib_ref] [bib_ref] Impact of graphene-based surfaces on the basic biological properties of human umbilical..., Jagiełło [/bib_ref] [bib_ref] Graphene nanomaterials: Synthesis, biocompatibility, and cytotoxicity, Liao [/bib_ref] [bib_ref] Nanostructured graphene surfaces promote different stages of bone cell differentiation, Borghi [/bib_ref] [bib_ref] Graphene family materials in bone tissue regeneration: Perspectives and challenges, Cheng [/bib_ref] [bib_ref] Osteogenic potential of graphene in bone tissue engineering scaffolds, Prasadh [/bib_ref] [bib_ref] Graphene: A versatile carbon-based material for bone tissue engineering, Dubey [/bib_ref] [bib_ref] Carbon nanostructures for hard tissue engineering, Han [/bib_ref]. For instance, MWNTs show potential applications as structural biomaterials and reinforcing nanofillers for degradable polymer scaffolds for bone tissue engineering [fig_ref] Figure 2: The design and fabrication of polyetheretherketone patient-specific implants using CAD/CAM system for... [/fig_ref] [bib_ref] Carbon nanotube scaffolds as emerging nanoplatform for myocardial tissue regeneration: A review..., Gorain [/bib_ref]. Graphene oxide (GO), a graphene derivative, with a Young's modulus of 207.6 ± 23.4 GPa [bib_ref] Mechanical properties of monolayer graphene oxide, Suk [/bib_ref] , also acting as an effective reinforcement for biodegradable polymer nanocomposites in bone tissue engineering [bib_ref] Antibacterial activities of aliphatic polyester nanocomposites with silver nanoparticles and/or graphene oxide..., Liao [/bib_ref] [bib_ref] Carbon nanotube scaffolds as emerging nanoplatform for myocardial tissue regeneration: A review..., Gorain [/bib_ref] [bib_ref] Multifunctional poly (glycolic acid-co-propylene fumarate) electrospun fibers reinforced with graphene oxide and..., Díez-Pascual [/bib_ref]. However, very scarce information is available in the literature relating the mechanical behavior and biocompatibility of PEEK/GO nanocomposites. Very recently, He et al. injection molded PEEK/(0.1-5 wt%)GO nanocomposites using PEEK powders and commercially available GO sheets [bib_ref] Super tough graphene oxide reinforced polyetheretherketone for potential hard tissue repair applications, He [/bib_ref]. The elastic modulus of pure PEEK is around 1.31 GPa, and remains unchanged by adding 0.5 wt% GO. The stiffness increases slightly to 1.34 GPa and 1.38 GPa by adding 1 and 2 wt% GO, respectively. Apparently, the use of raw PEEK powders would have little effect on the stiffness enhancement of PEEK/GO nanocomposites. [bib_ref] Direct mechanical measurement of the tensile strength and elastic modulus of multiwalled..., Demczyk [/bib_ref]. Thus, MWNTs are usually used to reinforce polymers for industrial and biomedical applications. Vapor grown carbon nanofiber (VGCNF) comprises of different arrangement of graphitic layers in herringbone and tubular features, with diameters ranging from 50 to 500 nm [bib_ref] Comparative study of herringbone and stacked-cup carbon nanofibers, Kim [/bib_ref] [bib_ref] Influence of structural and textural parameters of carbon nanofibers on their capacitive..., Moyseowicz [/bib_ref]. The mechanical properties of CNFs are inferior to those of MWNTs due to the presence of more crystalline defects. From biomedical perspective, carbonaceous nanomaterials promote the adhesion, growth and differentiation of bone cells [bib_ref] Applications of carbon nanotubes in bone regenerative medicine, Tanaka [/bib_ref] [bib_ref] Recent advances in the use of carbon nanotubes as smart biomaterials, Menezes [/bib_ref] [bib_ref] Cell attachment and spreading on carbon nanotubes is facilitated by integrin binding, Imaninezhad [/bib_ref] [bib_ref] Carbon nanostructures in bone tissue engineering, Perkins [/bib_ref] [bib_ref] Graphene-induced osteogenic differentiation is mediated by the integrin/FAK axis, Xie [/bib_ref] [bib_ref] Impact of graphene-based surfaces on the basic biological properties of human umbilical..., Jagiełło [/bib_ref] [bib_ref] Graphene nanomaterials: Synthesis, biocompatibility, and cytotoxicity, Liao [/bib_ref] [bib_ref] Nanostructured graphene surfaces promote different stages of bone cell differentiation, Borghi [/bib_ref] [bib_ref] Graphene family materials in bone tissue regeneration: Perspectives and challenges, Cheng [/bib_ref] [bib_ref] Osteogenic potential of graphene in bone tissue engineering scaffolds, Prasadh [/bib_ref] [bib_ref] Graphene: A versatile carbon-based material for bone tissue engineering, Dubey [/bib_ref] [bib_ref] Carbon nanostructures for hard tissue engineering, Han [/bib_ref]. For instance, MWNTs show potential applications as structural biomaterials and reinforcing nanofillers for degradable polymer scaffolds for bone tissue engineering [fig_ref] Figure 2: The design and fabrication of polyetheretherketone patient-specific implants using CAD/CAM system for... [/fig_ref] [bib_ref] Carbon nanotube scaffolds as emerging nanoplatform for myocardial tissue regeneration: A review..., Gorain [/bib_ref]. Graphene oxide (GO), a graphene derivative, with a Young's modulus of 207.6 ± 23.4 GPa [bib_ref] Mechanical properties of monolayer graphene oxide, Suk [/bib_ref] , also acting as an effective reinforcement for biodegradable polymer nanocomposites in bone tissue engineering [bib_ref] Antibacterial activities of aliphatic polyester nanocomposites with silver nanoparticles and/or graphene oxide..., Liao [/bib_ref] [bib_ref] Carbon nanotube scaffolds as emerging nanoplatform for myocardial tissue regeneration: A review..., Gorain [/bib_ref] [bib_ref] Multifunctional poly (glycolic acid-co-propylene fumarate) electrospun fibers reinforced with graphene oxide and..., Díez-Pascual [/bib_ref]. However, very scarce information is available in the literature relating the mechanical behavior and biocompatibility of PEEK/GO nanocomposites. Very recently, He et al. injection molded PEEK/(0.1-5 wt%)GO nanocomposites using PEEK powders and commercially available GO sheets [bib_ref] Super tough graphene oxide reinforced polyetheretherketone for potential hard tissue repair applications, He [/bib_ref]. The elastic modulus of pure PEEK is around 1.31 GPa, and remains unchanged by adding 0.5 wt% GO. The stiffness increases slightly to 1.34 GPa and 1.38 GPa by adding 1 and 2 wt% GO, respectively. Apparently, the use of raw PEEK powders would have little effect on the stiffness enhancement of PEEK/GO nanocomposites. Deng et al. fabricated PEEK/MWNT nanocomposites containing 6.5-15 wt% MWNTs using melt-extrusion and injection molding [bib_ref] Tensile properties at different temperature and observation of micro deformation of carbon..., Deng [/bib_ref]. The elastic modulus of injection molded PEEK increases from 4.0 GPa to 5.32 GPa, 6.35 GPa and 7.55 GPa by adding 6.5 wt%, 12 wt% and 15 wt% MWNT, respectively. The tensile strength of PEEK increases from 93.55 MPa to 102.15 MPa, 107.14 MPa and 110.90 MPa by adding 6.5 wt%, 12 wt% and 15 wt% MWNT, respectively. The fracture elongation of PEEK is greater than 20% and decreases to 8.28% and 6.28% by adding 12 wt% and 15 wt% MWNT, respectively. Apparently, the tensile characteristics of PEEK/15 wt% MWNTs meet the tensile properties of human cortical bone. The fracture elongation of this nanocomposite can maintain an adequate value of 6.28% due to the crack bridging effect of MWNTs of large aspect ratios, and Deng et al. fabricated PEEK/MWNT nanocomposites containing 6.5-15 wt% MWNTs using melt-extrusion and injection molding [bib_ref] Tensile properties at different temperature and observation of micro deformation of carbon..., Deng [/bib_ref]. The elastic modulus of injection molded PEEK increases from 4.0 GPa to 5.32 GPa, 6.35 GPa and 7.55 GPa by adding 6.5 wt%, 12 wt% and 15 wt% MWNT, respectively. The tensile strength of PEEK increases from 93.55 MPa to 102.15 MPa, 107.14 MPa and 110.90 MPa by adding 6.5 wt%, 12 wt% and 15 wt% MWNT, respectively. The fracture elongation of PEEK is greater than 20% and decreases to 8.28% and 6.28% by adding 12 wt% and 15 wt% MWNT, respectively. Apparently, the tensile characteristics of PEEK/15 wt% MWNTs meet the tensile properties of human cortical bone. The fracture elongation of this nanocomposite can maintain an adequate value of 6.28% due to the crack bridging effect of MWNTs of large aspect ratios, and excellent mechanical flexibility of nanotubes under the application of large deformation strains [bib_ref] Bending and buckling of carbon nanotubes under large strain, Falvo [/bib_ref]. Despite the beneficial effect of 15 wt% MWNT addition, the filler content is considered to be relatively high for a nanocomposite, which may induce filler aggregation.
Tjong and coworkers injection molded PEEK and its nanocomposites reinforced with 1.5 wt% MWNT and 3.0 wt% MWNT. Low loading levels of MWNTs were added to PEEK to prevent agglomeration of nanotubes [bib_ref] Polyetheretherketone hybrid composites with bioactive nanohydroxyapatite and multiwalled carbon nanotube fillers, Liu [/bib_ref]. The stiffness, tensile strength and elongation at break of PEEK/1.5 wt% MWNT are 4.21 ± 0.11 GPa, 83.38 ± 0.78 MPa and 57.25 ± 13.20%, respectively. Moreover, the stiffness, tensile strength and elongation at break of PEEK/3.0wt% MWNT are 4.25± 0.85 GPa, 82.08 ± 3.68 MPa, and 56.48 ± 24.90%, respectively. It is evident that the elastic modulus of PEEK/3.0 wt% MWNT nanocomposite is smaller than the low modulus range of cortical bone. However, the addition of 30 wt% nHA to this composite improves its stiffness to 7.13 ± 0.12 GPa, forming the so-called hybrid nanocomposite. [fig_ref] Figure 2: The design and fabrication of polyetheretherketone patient-specific implants using CAD/CAM system for... [/fig_ref] ,b are the SEM micrographs showing the attachment of murine MC3T3-E1 pre-osteoblasts on PEEK/3.0 wt% MWNT nanocomposite for two and four days, respectively. The adhesion of murine osteoblasts can be clearly seen on PEEK/1.5 wt% MWNT and PEEK/3.0 wt% MWNT composite surfaces by staining F-actin filaments and nuclei of osteoblasts [fig_ref] Figure 2: The design and fabrication of polyetheretherketone patient-specific implants using CAD/CAM system for... [/fig_ref]. It is evident that low loading levels of MWNTs promote the adhesion of osteoblasts on bioinert PEEK surface.
Polymers 2020, 8, x 24 of 51 excellent mechanical flexibility of nanotubes under the application of large deformation strains [bib_ref] Bending and buckling of carbon nanotubes under large strain, Falvo [/bib_ref].
Despite the beneficial effect of 15 wt% MWNT addition, the filler content is considered to be relatively high for a nanocomposite, which may induce filler aggregation. Tjong and coworkers injection molded PEEK and its nanocomposites reinforced with 1.5 wt% MWNT and 3.0 wt% MWNT. Low loading levels of MWNTs were added to PEEK to prevent agglomeration of nanotubes [bib_ref] Polyetheretherketone hybrid composites with bioactive nanohydroxyapatite and multiwalled carbon nanotube fillers, Liu [/bib_ref]. The stiffness, tensile strength and elongation at break of PEEK/1.5 wt% MWNT are 4.21 ± 0.11 GPa, 83.38 ± 0.78 MPa and 57.25 ± 13.20%, respectively. Moreover, the stiffness, tensile strength and elongation at break of PEEK/3.0wt% MWNT are 4.25± 0.85 GPa, 82.08 ± 3.68 MPa, and 56.48 ± 24.90%, respectively. It is evident that the elastic modulus of PEEK/3.0 wt% MWNT nanocomposite is smaller than the low modulus range of cortical bone. However, the addition of 30 wt% nHA to this composite improves its stiffness to 7.13 ± 0.12 GPa, forming the socalled hybrid nanocomposite. [fig_ref] Figure 2: The design and fabrication of polyetheretherketone patient-specific implants using CAD/CAM system for... [/fig_ref] ,b are the SEM micrographs showing the attachment of murine MC3T3-E1 pre-osteoblasts on PEEK/3.0 wt% MWNT nanocomposite for two and four days, respectively. The adhesion of murine osteoblasts can be clearly seen on PEEK/1.5 wt% MWNT and PEEK/3.0 wt% MWNT composite surfaces by staining F-actin filaments and nuclei of osteoblasts [fig_ref] Figure 2: The design and fabrication of polyetheretherketone patient-specific implants using CAD/CAM system for... [/fig_ref]. It is evident that low loading levels of MWNTs promote the adhesion of osteoblasts on bioinert PEEK surface. [bib_ref] Bending and buckling of carbon nanotubes under large strain, Falvo [/bib_ref]. Despite the beneficial effect of 15 wt% MWNT addition, the filler content is considered to be relatively high for a nanocomposite, which may induce filler aggregation. Tjong and coworkers injection molded PEEK and its nanocomposites reinforced with 1.5 wt% MWNT and 3.0 wt% MWNT. Low loading levels of MWNTs were added to PEEK to prevent agglomeration of nanotubes [bib_ref] Polyetheretherketone hybrid composites with bioactive nanohydroxyapatite and multiwalled carbon nanotube fillers, Liu [/bib_ref]. The stiffness, tensile strength and elongation at break of PEEK/1.5 wt% MWNT are 4.21 ± 0.11 GPa, 83.38 ± 0.78 MPa and 57.25 ± 13.20%, respectively. Moreover, the stiffness, tensile strength and elongation at break of PEEK/3.0wt% MWNT are 4.25± 0.85 GPa, 82.08 ± 3.68 MPa, and 56.48 ± 24.90%, respectively. It is evident that the elastic modulus of PEEK/3.0 wt% MWNT nanocomposite is smaller than the low modulus range of cortical bone. However, the addition of 30 wt% nHA to this composite improves its stiffness to 7.13 ± 0.12 GPa, forming the socalled hybrid nanocomposite. [fig_ref] Figure 2: The design and fabrication of polyetheretherketone patient-specific implants using CAD/CAM system for... [/fig_ref] ,b are the SEM micrographs showing the attachment of murine MC3T3-E1 pre-osteoblasts on PEEK/3.0 wt% MWNT nanocomposite for two and four days, respectively. The adhesion of murine osteoblasts can be clearly seen on PEEK/1.5 wt% MWNT and PEEK/3.0 wt% MWNT composite surfaces by staining F-actin filaments and nuclei of osteoblasts [fig_ref] Figure 2: The design and fabrication of polyetheretherketone patient-specific implants using CAD/CAM system for... [/fig_ref]. It is evident that low loading levels of MWNTs promote the adhesion of osteoblasts on bioinert PEEK surface. Fluorescence micrographs of murine MC3T3-E1 pre-osteoblasts cultivated on (a) PEEK/1.5 wt% MWNT and (b) PEEK/3.0 wt% MWNT nanocomposites for 3 days, and stained with F-actin cytoskeleton (green) and nucleus (blue). Reproduced from [bib_ref] Polyetheretherketone hybrid composites with bioactive nanohydroxyapatite and multiwalled carbon nanotube fillers, Liu [/bib_ref] with permission of MDPI.
## Hybrid composites
From the above discussion, it appears that the reinforcement made of single type of material is insufficient to achieve desired biocompatibility and mechanical properties of polymer composites for load-bearing applications. The filler reinforcement is the load-carrying component of the composites, while the matrix transfers the applied load to the fillers during mechanical testing. For instance, the elastic modulus, tensile strength, and fracture strain of injection molded PEEK/30 wt% SCF composite meet the tensile property requirement of cortical bone. However, this composite lacks bioactive fillers such as nHA for promoting adhesion, growth and differentiation of osteoblasts on its surface [bib_ref] Use of a novel carbon fibre composite material for the femoral stem..., Scotchford [/bib_ref]. As aforementioned, the elastic modulus of PEEK/3.0 wt% MWNT is 4.25 ± 0.85 GPa, i.e., smaller than the low modulus range of cortical bone. It can reach 7.13 ± 0.12 GPa by adding an optimal amount of nHA [bib_ref] Polyetheretherketone hybrid composites with bioactive nanohydroxyapatite and multiwalled carbon nanotube fillers, Liu [/bib_ref]. In this respect, hybridization of fillers of different materials is considered to be effective to obtain desired mechanical stiffness and biocompatibility of resulting biocomposites [bib_ref] Preparation, characterization, cellular response and in vivo osseointegration of polyetheretherketone/nano-hydroxyapatite/carbon fiber ternary..., Deng [/bib_ref].
## Peek-nha-mwnt hybrids
Tjong and coworkers explored the stiffness enhancement of PEEK/30 wt% nHA by adding low loading levels of CNFs or MWNTs. The results are listed in [fig_ref] Table 2: Tensile properties of PEEK-based nanocomposites and hybrids [/fig_ref] [bib_ref] Preparation of polyetheretherketone composites with nanohydroxyapatite rods and carbon nanofibers having high..., Chan [/bib_ref] [bib_ref] Polyetheretherketone hybrid composites with bioactive nanohydroxyapatite and multiwalled carbon nanotube fillers, Liu [/bib_ref]. For the purposes of comparison, this Table also summarizes the tensile properties of PEEK-based nanocomposites. By adding 3.0 wt% MWNT to PEEK/30wt% nHA, the hybrid composite exhibits a stiffness of 7.13 ± 0.12 GPa, tensile strength of 64.48 ± 8.51 MPa and tensile elongation of 1.74 ± 0.58%. Thus hybridizing bioactive nHA with MWNTs can produce PEEK-based hybrid with elastic modulus close to that of cortical bone. [fig_ref] Figure 2: The design and fabrication of polyetheretherketone patient-specific implants using CAD/CAM system for... [/fig_ref] shows that the inclusions of 1.5 wt% and 3.0 wt% MWNT to hydrophobic PEEK have little effect in reducing the water contact angles. However, the additions of 1.5 wt% and 3.0 wt% MWNT to PEEK/30 wt% nHA composite lead to a large drop in water contact angle values. The water contact angle declines markedly to 84.08 - and 76.99 - by hybridizing 30 wt%nHA with 1.5 wt% MWNT and 3.0 wt% MWNT. So a synergistic effect exists between nHA and MWNT in converting hydrophobic PEEK to hydrophilic. Due to improved surface wettability, hydrophilic PEEK/nHA-MWNT hybrids favor cellular adhesion. This is manifested by enhanced proliferation, differentiation and mineralization of murine MC3T3-E1 cells on the hybrid composite surfaces as revealed by MTT, ALP activity and Alizarin red-S staining results [fig_ref] Figure 2: The design and fabrication of polyetheretherketone patient-specific implants using CAD/CAM system for... [/fig_ref]. The large increase in ALP activity level of PEEK/30 wt%nHA-1.5 wt% MWNT and PEEK/30 wt%nHA-3.0 wt%MWNT hybrids at day 7 shows good indication of osteoblastic differentiation. Nowadays, the emergence and increasing use of nanomaterials have raised the concerns about their potential health hazards. CNTs have emerged as a new tool for transporting and delivering drug molecules [bib_ref] Single-walled carbon nanotube-assisted antibiotic delivery and imaging in S. epidermidis strains addressing..., Khazi-Syed [/bib_ref]. However, CNTs with large aspect ratios and surface areas as well as needlelike features would cause damages to human organs such as skins, lungs, kidneys, brains, etc. Possible entry routes include dermal contact, inhalation, injection and digestion during manufacturing or biomedical use. In vitro and in vivo studies have reported that CNTs can induce cytotoxicity via the generation of ROS and oxidative stress [bib_ref] In vivo toxicity assessment of occupational components of the carbon nanotube life..., Bishop [/bib_ref] [bib_ref] The effects of varying degree of MWCNT carboxylation on bioactivity in various..., Hamilton [/bib_ref] [bib_ref] The toxicity and therapeutic effects of single-and multi-wall carbon nanotubes on mice..., Kavosi [/bib_ref]. From in vivo animal models, needle-like CNTs are trapped by the lung, liver, spleen, etc. upon exposure to nanotubes. CNTs then penetrate the cell wall and reside in the cytoplasm of target organ cells [bib_ref] Biodistribution of carbon nanotubes in animal models, Jacobsen [/bib_ref]. It is noted those studies Nowadays, the emergence and increasing use of nanomaterials have raised the concerns about their potential health hazards. CNTs have emerged as a new tool for transporting and delivering drug molecules [bib_ref] Single-walled carbon nanotube-assisted antibiotic delivery and imaging in S. epidermidis strains addressing..., Khazi-Syed [/bib_ref]. However, CNTs with large aspect ratios and surface areas as well as needle-like features would cause damages to human organs such as skins, lungs, kidneys, brains, etc. Possible entry routes include dermal contact, inhalation, injection and digestion during manufacturing or biomedical use. In vitro and in vivo studies have reported that CNTs can induce cytotoxicity via the generation of ROS and oxidative stress [bib_ref] In vivo toxicity assessment of occupational components of the carbon nanotube life..., Bishop [/bib_ref] [bib_ref] The effects of varying degree of MWCNT carboxylation on bioactivity in various..., Hamilton [/bib_ref] [bib_ref] The toxicity and therapeutic effects of single-and multi-wall carbon nanotubes on mice..., Kavosi [/bib_ref]. From in vivo animal models, needle-like CNTs are trapped by the lung, liver, spleen, etc. upon exposure to nanotubes. CNTs then penetrate the cell wall and reside in the cytoplasm of target organ cells [bib_ref] Biodistribution of carbon nanotubes in animal models, Jacobsen [/bib_ref]. It is noted those studies were carried out under a direct contact of cell lines or animals with standalone carbon nanotubes. In polymer nanocomposites, CNT fillers are encapsulated and embedded firmly in the polymer matrix, thereby diminishing their toxic effects [bib_ref] The development, fabrication and material characterization of polypropylene composites reinforced with carbon..., Liao [/bib_ref] [bib_ref] Preparation of polyetheretherketone composites with nanohydroxyapatite rods and carbon nanofibers having high..., Chan [/bib_ref] [bib_ref] Polyetheretherketone hybrid composites with bioactive nanohydroxyapatite and multiwalled carbon nanotube fillers, Liu [/bib_ref].
## Peek-nha-cf hybrids
As mentioned, the tensile properties of injection molded PEEK/30 wt% SCF composite are similar to those of cortical bone [bib_ref] Use of a novel carbon fibre composite material for the femoral stem..., Scotchford [/bib_ref]. This microcomposite exhibits a poor ability to induce osteoblastic cell adhesion and proliferation. The biocompatibility of PEEK/CF composite can be improved by plasma spraying of nHA on its surface. However, the high-temperature environment of plasma sprayed gun would degrade nHA during deposition [bib_ref] Plasma-sprayed Hydroxyapatite-based coatings: Chemical, mechanical, microstructural, and biomedical properties, Heimann [/bib_ref]. Alternatively, nHA can be used as a bioactive filler for PEEK/CF composites. Recently, Wei and coworkers fabricated PEEK/25wt%nHA-20 wt%SCF via injection molding. The length and diameter of SCFs were 109 µm and 4 µm, respectively. The elastic modulus of PEEK was 3.5 ± 0.3 GPa, and reached 16.5 ± 0.7 GPa by adding 25 wt%nHA and 20 wt%SCF [bib_ref] Preparation, characterization, cellular response and in vivo osseointegration of polyetheretherketone/nano-hydroxyapatite/carbon fiber ternary..., Deng [/bib_ref]. The addition of 25 wt%nHA to PEEK/20 wt%SCF promotes the adhesion and growth of MG63 osteoblasts on its surface. After proliferation, the cells begin to differentiate by secreting bone matrix proteins. [fig_ref] Figure 2: The design and fabrication of polyetheretherketone patient-specific implants using CAD/CAM system for... [/fig_ref] shows the ALP activity of MG63 cells cultured on PEEK/25 wt%nHA-20 wt%SCF for different time points. At day 7, pure PEEK and hybrid composite show a similar ALP activity level. At day 17, the ALP activity level of both samples increases in which the hybrid showing a higher activity. Meanwhile, MG63 cells begin to mineralize the ECM at this stage [fig_ref] Figure 2: The design and fabrication of polyetheretherketone patient-specific implants using CAD/CAM system for... [/fig_ref]. The ALP activity declines on both samples at day 21, which marks the final phase of differentiation. At this time point, the mineralization level on the hybrid is increased largely compared to pure PEEK. [fig_ref] Figure 2: The design and fabrication of polyetheretherketone patient-specific implants using CAD/CAM system for... [/fig_ref] shows the formation of mineralized nodules due to osteoblasts on both sample surfaces at day 14 and day 21 as revealed by Alizarin red staining. These results demonstrate that the nHA filler of hybrid composite enhances the mineralization of ECM, thus facilitating the bone formation. In this respect, PEEK/25 wt%nHA-20 wt%SCF hybrid implants are inserted into tooth defects of male beagle dogs by extracting the rear molars in the upper and lower jaws. The hybrid composite implant exhibits higher osseointegration than PEEK based on 3D microcomputed tomography and histological analysis of bone ingrowth in the implants. So, the PEEK/25 wt%nHA-20 wt%SCF hybrid shows potential application for fabricating dental implants. were carried out under a direct contact of cell lines or animals with standalone carbon nanotubes. In polymer nanocomposites, CNT fillers are encapsulated and embedded firmly in the polymer matrix, thereby diminishing their toxic effects [bib_ref] The development, fabrication and material characterization of polypropylene composites reinforced with carbon..., Liao [/bib_ref] [bib_ref] Preparation of polyetheretherketone composites with nanohydroxyapatite rods and carbon nanofibers having high..., Chan [/bib_ref] [bib_ref] Polyetheretherketone hybrid composites with bioactive nanohydroxyapatite and multiwalled carbon nanotube fillers, Liu [/bib_ref].
## Peek-nha-cf hybrids
As mentioned, the tensile properties of injection molded PEEK/30 wt% SCF composite are similar to those of cortical bone [bib_ref] Use of a novel carbon fibre composite material for the femoral stem..., Scotchford [/bib_ref]. This microcomposite exhibits a poor ability to induce osteoblastic cell adhesion and proliferation. The biocompatibility of PEEK/CF composite can be improved by plasma spraying of nHA on its surface. However, the high-temperature environment of plasma sprayed gun would degrade nHA during deposition [bib_ref] Plasma-sprayed Hydroxyapatite-based coatings: Chemical, mechanical, microstructural, and biomedical properties, Heimann [/bib_ref]. Alternatively, nHA can be used as a bioactive filler for PEEK/CF composites. Recently, Wei and coworkers fabricated PEEK/25wt%nHA-20 wt%SCF via injection molding. The length and diameter of SCFs were 109 µm and 4 µm, respectively. The elastic modulus of PEEK was 3.5 ± 0.3 GPa, and reached 16.5 ± 0.7 GPa by adding 25 wt%nHA and 20 wt%SCF [bib_ref] Preparation, characterization, cellular response and in vivo osseointegration of polyetheretherketone/nano-hydroxyapatite/carbon fiber ternary..., Deng [/bib_ref]. The addition of 25 wt%nHA to PEEK/20 wt%SCF promotes the adhesion and growth of MG63 osteoblasts on its surface. After proliferation, the cells begin to differentiate by secreting bone matrix proteins. [fig_ref] Figure 2: The design and fabrication of polyetheretherketone patient-specific implants using CAD/CAM system for... [/fig_ref] shows the ALP activity of MG63 cells cultured on PEEK/25 wt%nHA-20 wt%SCF for different time points. At day 7, pure PEEK and hybrid composite show a similar ALP activity level. At day 17, the ALP activity level of both samples increases in which the hybrid showing a higher activity. Meanwhile, MG63 cells begin to mineralize the ECM at this stage [fig_ref] Figure 2: The design and fabrication of polyetheretherketone patient-specific implants using CAD/CAM system for... [/fig_ref]. The ALP activity declines on both samples at day 21, which marks the final phase of differentiation. At this time point, the mineralization level on the hybrid is increased largely compared to pure PEEK. [fig_ref] Figure 2: The design and fabrication of polyetheretherketone patient-specific implants using CAD/CAM system for... [/fig_ref] shows the formation of mineralized nodules due to osteoblasts on both sample surfaces at day 14 and day 21 as revealed by Alizarin red staining. These results demonstrate that the nHA filler of hybrid composite enhances the mineralization of ECM, thus facilitating the bone formation. In this respect, PEEK/25 wt%nHA-20 wt%SCF hybrid implants are inserted into tooth defects of male beagle dogs by extracting the rear molars in the upper and lower jaws. The hybrid composite implant exhibits higher osseointegration than PEEK based on 3D microcomputed tomography and histological analysis of bone ingrowth in the implants. So, the PEEK/25 wt%nHA-20 wt%SCF hybrid shows potential application for fabricating dental implants.
## Scaffolds for soft tissue engineering
Synthetic polymer scaffolds have been employed in tissue engineering to offer a structurally stable environment for bone regeneration. A scaffold serves as an artificial ECM that mimics the structure and function of natural bone. So, the scaffold should possess a 3D, interconnected porous structure that provides a structural support for the adhesion, growth and differentiation of The quantitative value of matrix mineralization acquired by measuring optical density. (c) Alizarin red staining for calcium-rich deposits secreted by MG63 cells on PEEK and PEEK/nHA-SCF hybrid. * represents p < 0.05 and ** indicates p < 0.01 compared with PEEK/nHA-SCF. Reproduced from [bib_ref] Preparation, characterization, cellular response and in vivo osseointegration of polyetheretherketone/nano-hydroxyapatite/carbon fiber ternary..., Deng [/bib_ref] with permission of Elsevier.
## Scaffolds for soft tissue engineering
Synthetic polymer scaffolds have been employed in tissue engineering to offer a structurally stable environment for bone regeneration. A scaffold serves as an artificial ECM that mimics the structure and function of natural bone. So, the scaffold should possess a 3D, interconnected porous structure that provides a structural support for the adhesion, growth and differentiation of osteoblasts. As such, the scaffold for bone tissue engineering should provide osteoinductive, osteoconductive, and osteointegrative properties [bib_ref] Polymer-based scaffolds for soft-tissue engineering, Perez-Puyana [/bib_ref] [bib_ref] Fabrication of scaffolds for bone-tissue regeneration, Chocholata [/bib_ref] [bib_ref] Novel 3D hybrid nanofiber scaffolds for bone regeneration, Kołbuk [/bib_ref] [bib_ref] Bone regeneration based on tissue engineering conceptions-A 21st century perspective, Henkel [/bib_ref]. Human bone tissues comprise of dense cortical bone and porous cancellous/trabecular bone having a porosity of 50-90%. The design of polymer scaffolds concentrates on mimicking cancellous bone structure. The porosity of scaffolds is designed to make them resemble cancellous bone for tissue regeneration. Biodegradable natural and synthetic polymers are typical materials used for constructing porous scaffolds [bib_ref] Polymer-and hybrid-based biomaterials for interstitial, connective, vascular, nerve, visceral and musculoskeletal tissue..., Abalymov [/bib_ref] [bib_ref] Solvent-free approaches for the processing of scaffolds in regenerative medicine, Santos-Rosales [/bib_ref] [bib_ref] Natural and synthetic polymers for bone scaffolds optimization, Donnaloja [/bib_ref].
Porous polymer scaffolds can be fabricated from several techniques such as solvent casting-particulate leaching, gas foaming, freeze drying, thermally induced phase separation, electrospinning and 3D printing. These techniques have their own advantages and shortcomings [bib_ref] Fabrication of scaffolds for bone-tissue regeneration, Chocholata [/bib_ref]. For instance, solvent casting/particulate leaching method involves an initial dissolution of polymer in an organic solvent, followed by porogens mixing. The last step procedures include mold casting and water immersion for leaching porogens. The main limitations of this technique include the use of a toxic solvent for the polymer dissolution, prolonged water soaking, and a small membrane thickness. Electrospinning can produce ECM-mimicking fibrous network with high porosity and interconnected pores. However, the pores generated are rather small, thus preventing the ingrowth of bone cells. In recent years, 3D-printing techniques such as FFF, stereolithography (SLA) and selective laser sintering (SLS) have been used increasingly for printing 3D porous scaffolds for bone tissue engineering [bib_ref] Fabrication of scaffolds for bone-tissue regeneration, Chocholata [/bib_ref] [bib_ref] Current advances and future perspectives in extrusion-based bioprinting, Ozbolat [/bib_ref] [bib_ref] Polymer-ceramic composite scaffolds: The effect of hydroxyapatite and β-tricalcium phosphate, Huang [/bib_ref] [bib_ref] Extrusion-based additive manufacturing of PEEK for biomedical applications, Vaezi [/bib_ref] [bib_ref] Additively-manufactured poly-ether-ether-ketone (PEEK) lattice scaffolds with uniform microporous architectures for enhanced cellular..., Su [/bib_ref] [bib_ref] Three-dimensionally-printed polyether-ether-ketone implant with a cross-linked structure and acid-etched microporous surface promotes..., Feng [/bib_ref]. AM technology is very effective to fabricate porous polymer scaffolds with the accurate control of the pore size, porosity and interconnected pore network. Thus, the printed 3D scaffolds are capable of sustaining vascularization, cell transport and tissue ingrowth [bib_ref] Bioprinting for vascular and vascularized tissue biofabrication, Datta [/bib_ref].
PEEK is a semicrystalline polymer with a high melting temperature of 340 - C [bib_ref] High-performance nanocomposites based on polyetherketones, Díez-Pascual [/bib_ref]. Because of its high melting temperature, it is more difficult to print 3D PEEK products than other thermoplastics. Nevertheless, some workers have successfully printed porous PEEK scaffolds by monitoring the printing parameters properly [bib_ref] Extrusion-based additive manufacturing of PEEK for biomedical applications, Vaezi [/bib_ref] [bib_ref] Additively-manufactured poly-ether-ether-ketone (PEEK) lattice scaffolds with uniform microporous architectures for enhanced cellular..., Su [/bib_ref] [bib_ref] Three-dimensionally-printed polyether-ether-ketone implant with a cross-linked structure and acid-etched microporous surface promotes..., Feng [/bib_ref]. Very recently, Su et al. employed FFF technique to print 3D porous scaffolds followed by heat treatment and sulfonation. The FFF-printed PEEK scaffolds were heat treated at 200 - C for 1.5 h and then at 300 - C for another 1.5 h to improve the mechanical properties. Subsequently, micropores were induced on the PEEK filaments of heat-treated scaffolds via sulfonation in concentrated sulfuric acid [fig_ref] Figure 2: The design and fabrication of polyetheretherketone patient-specific implants using CAD/CAM system for... [/fig_ref] [bib_ref] Additively-manufactured poly-ether-ether-ketone (PEEK) lattice scaffolds with uniform microporous architectures for enhanced cellular..., Su [/bib_ref]. Accordingly, bone cells can attach readily onto rough PEEK filaments prior to cellular ingrowth through the large pores created by FFF. [fig_ref] Figure 2: The design and fabrication of polyetheretherketone patient-specific implants using CAD/CAM system for... [/fig_ref] shows the proliferation of MC3T3-E1 cells cultured on pure PEEK, heat-treated PEEK (HPEEK) and sulfonated PEEK (SHPEEK) scaffolds for different time points as determined by Cell Counting Kit-8 assay. At day 3, there exists no significant difference in the cell viability between PEEK and HPEEK scaffolds. In contrast, the presence of uniform micropores on SHPEEK scaffold promotes better cellular adhesion and proliferation after culturing for 3 and 5 days. [fig_ref] Figure 3: Current strategies typically used to improve bioactivity of PEEK [/fig_ref] -d show the mineralization of ECM by MC3T3-E1 cells cultured on different scaffolds for 7days. Very few calcified nodules are deposited on the surface filaments of PEEK and HPEEK scaffolds. However, the filaments of SHPEEK scaffold are completely stained with a red dye, demonstrating the abundant deposition of calcified nodules on the filaments. Thus, micropores created on the PEEK filaments provides the sites for osteoblastic adhesion, proliferation and mineralization.
(HPEEK) and sulfonated PEEK (SHPEEK) scaffolds for different time points as determined by Cell Counting Kit-8 assay. At day 3, there exists no significant difference in the cell viability between PEEK and HPEEK scaffolds. In contrast, the presence of uniform micropores on SHPEEK scaffold promotes better cellular adhesion and proliferation after culturing for 3 and 5 days. [fig_ref] Figure 3: Current strategies typically used to improve bioactivity of PEEK [/fig_ref] -d show the mineralization of ECM by MC3T3-E1 cells cultured on different scaffolds for 7days. Very few calcified nodules are deposited on the surface filaments of PEEK and HPEEK scaffolds. However, the filaments of SHPEEK scaffold are completely stained with a red dye, demonstrating the abundant deposition of calcified nodules on the filaments. Thus, micropores created on the PEEK filaments provides the sites for osteoblastic adhesion, proliferation and mineralization. deposition of calcified nodules on the filaments. Thus, micropores created on the PEEK filaments provides the sites for osteoblastic adhesion, proliferation and mineralization.
## Biodegradable peek blend scaffolds
The use of PEEK as a biomaterial to make scaffolds in soft tissue engineering is hampered by its non-degradation behavior. PEEK is hydrophobic, exhibiting no degradation upon exposure in aqueous environments. On the other hand, biodegradable polymers such as polyglycolic acid (PGA) and polyvinyl alcohol (PVA) can be blended with PEEK to produce degradable scaffolds [bib_ref] Polyetheretherketone/poly (glycolic acid) blend scaffolds with biodegradable properties, Shuai [/bib_ref] [bib_ref] Characterization and bioactivity evaluation of (polyetheretherketone/polyglycolicacid)-hydroyapatite scaffolds for tissue regeneration, Shuai [/bib_ref] [bib_ref] Graphene oxide-driven interfacial coupling in laser 3D printed PEEK/PVA scaffolds for bone..., Feng [/bib_ref]. PGA is an aliphatic polyester showing very fast degradation in aqueous solutions due to its hydrolytic nature. PGA decomposes into glycolic acid through ester hydrolysis. The acidic product further degrades the polymer in an autocatalytic effect [bib_ref] Hydrolytic degradation and erosion of polyester biomaterials, Woodard [/bib_ref]. PGA is often used as a suture material clinically. However, the fast degradation rate and poor mechanical strength limit its application in tissue engineering [bib_ref] Evaluation of tensile strength of surgical synthetic absorbable suture materials: An in..., Khiste [/bib_ref]. By combining PEEK with PGA, the resulting PEEK/PGA blends are able to degrade; the degradation rate can be tuned by monitoring the PGA content of PEEK/PGA blends. In addition, PEEK with high stiffness and strength can sustain the structure of PEEK/PGA scaffolds from collapsing during the degradation of PGA in aqueous environments.
More Reproduced from [bib_ref] Additively-manufactured poly-ether-ether-ketone (PEEK) lattice scaffolds with uniform microporous architectures for enhanced cellular..., Su [/bib_ref] with permission of Elsevier under a Creative Commons license.
## Biodegradable peek blend scaffolds
The use of PEEK as a biomaterial to make scaffolds in soft tissue engineering is hampered by its non-degradation behavior. PEEK is hydrophobic, exhibiting no degradation upon exposure in aqueous environments. On the other hand, biodegradable polymers such as polyglycolic acid (PGA) and polyvinyl alcohol (PVA) can be blended with PEEK to produce degradable scaffolds [bib_ref] Polyetheretherketone/poly (glycolic acid) blend scaffolds with biodegradable properties, Shuai [/bib_ref] [bib_ref] Characterization and bioactivity evaluation of (polyetheretherketone/polyglycolicacid)-hydroyapatite scaffolds for tissue regeneration, Shuai [/bib_ref] [bib_ref] Graphene oxide-driven interfacial coupling in laser 3D printed PEEK/PVA scaffolds for bone..., Feng [/bib_ref]. PGA is an aliphatic polyester showing very fast degradation in aqueous solutions due to its hydrolytic nature. PGA decomposes into glycolic acid through ester hydrolysis. The acidic product further degrades the polymer in an autocatalytic effect [bib_ref] Hydrolytic degradation and erosion of polyester biomaterials, Woodard [/bib_ref]. PGA is often used as a suture material clinically. However, the fast degradation rate and poor mechanical strength limit its application in tissue engineering [bib_ref] Evaluation of tensile strength of surgical synthetic absorbable suture materials: An in..., Khiste [/bib_ref]. By combining PEEK with PGA, the resulting PEEK/PGA blends are able to degrade; the degradation rate can be tuned by monitoring the PGA content of PEEK/PGA blends. In addition, PEEK with high stiffness and strength can sustain the structure of PEEK/PGA scaffolds from collapsing during the degradation of PGA in aqueous environments.
More recently, Shuai et al. fabricated porous PEEK/PGA blend scaffolds containing 20 wt% and 40 wt% PGA using selective laser sintering (SLS) process [bib_ref] Polyetheretherketone/poly (glycolic acid) blend scaffolds with biodegradable properties, Shuai [/bib_ref]. SLS is one of the AM techniques employing a laser beam to sinter polymer powder materials in a layer by layer to construct 3D scaffolds. [fig_ref] Figure 1: Multiple stages involved in the assessment of newly developed biomaterials for bone... [/fig_ref] shows the compressive strength and tensile strength vs PGA content for SLS-printed PEEK/PGA scaffolds. Both mechanical strengths decrease with increasing PGA content as expected. The compressive strength of printed PEEK and PEEK/PGA scaffolds is higher than that of cancellous bone ranging from 2-12 MPa. [fig_ref] Figure 1: Multiple stages involved in the assessment of newly developed biomaterials for bone... [/fig_ref] shows the weight loss vs time plots of PEEK, PEEK/20 wt% PGA and PEEK/4 0wt% PGA scaffolds upon immersion in a simulated body fluid (SBF) at 37 - C for different periods. The PEEK scaffold exhibits no weight loss after immersion in SBF for up to 28 days. However, PEEK/PGA scaffolds show distinct degradation behavior in SBF as revealed by a marked increase in the weight loss with immersion time. The weight loss of the scaffolds increases with increasing PGA content in the polymer blend. The water uptake into hydrolytic PGA phase leads to its dissolution in SBF. Consequently, degradation-induced pores are found on the surfaces of PEEK/PGA blend scaffolds. PEEK and PEEK-PGA scaffolds are bioinert, thus their surfaces are unfavorable for osteoblastic adhesion and proliferation. The scaffolds must achieve good compatibility with the surrounding tissue to ensure bone regeneration. In general, SLS-built scaffolds have rough surfaces, contributing somewhat to bone cell adhesion. So sulfonation is unnecessary for creating micropores on the PEEK filaments of scaffolds for cell attachment. The biocompatibility of SLS-printed PEEK and PEEK-PGA scaffolds can be further improved by adding nHA fillers. In another study, Shuai et al. [fig_ref] Figure 3: Current strategies typically used to improve bioactivity of PEEK [/fig_ref] ,b. The pH of PBS decreases with immersion time due to the release of glycolic acid byproduct. The 10%nHA filler of the PEEK-20%PGA/10%nHA scaffold neutralizes autocatalytic effect due to acidic PGA byproduct. This is beneficial in reducing inflammation of wounds. So, PEEK-20%PGA scaffold exhibits a higher weight loss at different time points compared to PEEK-20%PGA/10%nHA due to the autocatalytic mechanism [bib_ref] Hydrolytic degradation and erosion of polyester biomaterials, Woodard [/bib_ref]. The nHA filler favors the adhesion and proliferation of MG63 cells on the surface of PEEK-20%PGA/10%nHA scaffold [fig_ref] Figure 3: Current strategies typically used to improve bioactivity of PEEK [/fig_ref]. The [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] MTT cell proliferation assay results also reveal that the cell viability of PEEK-20%PGA/10%nHA is higher than that of PEEK-20%PGA. Finally, ALP activity level of MG63 cells cultured on the PEEK-20%PGA/10%nHA scaffold is considerably higher than that on PEEK-20%PGA scaffold. As mentioned above, GO additions show little enhancement in stiffness of injection molded PEEK/GO nanocomposites [bib_ref] Super tough graphene oxide reinforced polyetheretherketone for potential hard tissue repair applications, He [/bib_ref]. On the contrary, GO sheets can be dispersed homogeneously in the polymeric matrix using solution mixing process. In this respect, hydrolytic PVA, GO and PEEK are dispersed and mixed in water under sonication, followed by filtering and drying. The laser beam of As mentioned above, GO additions show little enhancement in stiffness of injection molded PEEK/GO nanocomposites [bib_ref] Super tough graphene oxide reinforced polyetheretherketone for potential hard tissue repair applications, He [/bib_ref]. On the contrary, GO sheets can be dispersed homogeneously in the polymeric matrix using solution mixing process. In this respect, hydrolytic PVA, GO and PEEK are dispersed and mixed in water under sonication, followed by filtering and drying. The laser beam of As mentioned above, GO additions show little enhancement in stiffness of injection molded PEEK/GO nanocomposites [bib_ref] Super tough graphene oxide reinforced polyetheretherketone for potential hard tissue repair applications, He [/bib_ref]. On the contrary, GO sheets can be dispersed homogeneously in the polymeric matrix using solution mixing process. In this respect, hydrolytic PVA, GO and PEEK are dispersed and mixed in water under sonication, followed by filtering and drying. The laser beam of As mentioned above, GO additions show little enhancement in stiffness of injection molded PEEK/GO nanocomposites [bib_ref] Super tough graphene oxide reinforced polyetheretherketone for potential hard tissue repair applications, He [/bib_ref]. On the contrary, GO sheets can be dispersed homogeneously in the polymeric matrix using solution mixing process. In this respect, hydrolytic PVA, GO and PEEK are dispersed and mixed in water under sonication, followed by filtering and drying. The laser beam of SLS is then used to sinter mixed PEEK-PVA/GO powders to form 3D porous scaffolds. The mass ratio of PEEK to PVA is fixed at 7:3 (wt/wt) [fig_ref] Figure 3: Current strategies typically used to improve bioactivity of PEEK [/fig_ref] [bib_ref] Graphene oxide-driven interfacial coupling in laser 3D printed PEEK/PVA scaffolds for bone..., Feng [/bib_ref]. The additions of 0.5-2 wt% GO to PEEK-PVA blend enhance the compressive strength and compressive modules markedly, especially at 1wt% GO [fig_ref] Figure 3: Current strategies typically used to improve bioactivity of PEEK [/fig_ref]. These results reveal remarkable strengthening and stiffening effects of GO nanofillers. The compressive strength and compressive modulus of PEEK-PVA scaffold are determined to be 10.12 MPa and 1.22 GPa, respectively; while those of PEEK-PVA/1 wt% GO scaffold are 20.13 MPa and 1.82 GPa, respectively. So, the compressive strength of PEEK-PVA/1 wt% GO scaffold is twice higher than that of PEEK-PVA scaffold. Furthermore, the compressive strength of PEEK-PVA/1 wt% GO scaffold is considerably higher than that of cancellous bone ranging from 2-12 MPa. From the literature, the compressive modulus of wet human cancellous bone and dry bovine cancellous bone is 0.44 ± 0.27 GPa and 1.4 ± 0.3 GPa, respectively [bib_ref] Recent advances on the measurement and calculation of the elastic moduli of..., Novitskaya [/bib_ref]. The compressive modulus of PEEK-PVA/1 wt% GO scaffold exceeds that of mammalian cancellous bone. So, this composite scaffold with enhanced compressive strength and stiffness can provide a strong support for bone cell adhesion, proliferation and bone tissue regeneration. It is well known that GO is hydrophilic due to the presence of oxygenated functional groups. PEEK is hydrophobic, and the water contact angle reduces to 85.52° by blending with hydrolytic PVA. The water contact angle further reduces to 78.16° by adding 1wt% GO to PEEK-PVA. As a result, the water absorption of PEEK-PVA/1wt% GO scaffold increases with immersion time in PBS at 37 °C, leading to the dissolution of PVA phase and weight loss increment accordingly. Some pores are formed on the scaffold surface due to the PVA degradation, and the number of pores increases with immersion time [fig_ref] Figure 3: Current strategies typically used to improve bioactivity of PEEK [/fig_ref]. The GO sheets are known to promote the adhesion, growth and differentiation of osteoblasts [bib_ref] Graphene nanomaterials: Synthesis, biocompatibility, and cytotoxicity, Liao [/bib_ref] [bib_ref] Phosphate graphene as an intrinsically osteoinductive scaffold for stem cell-driven bone regeneration, Arnold [/bib_ref] [bib_ref] Graphene oxide promotes embryonic stem cell differentiation to haematopoietic lineage, Garcia-Alegria [/bib_ref]. Therefore, PEEK-PVA/1 wt% GO scaffold promotes the attachment, proliferation and differentiation of MG63 cells as revealed by live/dead assay and CCK-8 assay results and encourages new bone formation in New Zealand white rabbits in vivo. [fig_ref] Table 3: The strengths and weaknesses of 3D-printed PEEK-based scaffolds [/fig_ref] lists the strengths and weaknesses of 3D-printed PEEK-based scaffolds prepared by The additions of 0.5-2 wt% GO to PEEK-PVA blend enhance the compressive strength and compressive modules markedly, especially at 1wt% GO [fig_ref] Figure 3: Current strategies typically used to improve bioactivity of PEEK [/fig_ref]. These results reveal remarkable strengthening and stiffening effects of GO nanofillers. The compressive strength and compressive modulus of PEEK-PVA scaffold are determined to be 10.12 MPa and 1.22 GPa, respectively; while those of PEEK-PVA/1 wt% GO scaffold are 20.13 MPa and 1.82 GPa, respectively. So, the compressive strength of PEEK-PVA/1 wt% GO scaffold is twice higher than that of PEEK-PVA scaffold. Furthermore, the compressive strength of PEEK-PVA/1 wt% GO scaffold is considerably higher than that of cancellous bone ranging from 2-12 MPa. From the literature, the compressive modulus of wet human cancellous bone and dry bovine cancellous bone is 0.44 ± 0.27 GPa and 1.4 ± 0.3 GPa, respectively [bib_ref] Recent advances on the measurement and calculation of the elastic moduli of..., Novitskaya [/bib_ref]. The compressive modulus of PEEK-PVA/1 wt% GO scaffold exceeds that of mammalian cancellous bone. So, this composite scaffold with enhanced compressive strength and stiffness can provide a strong support for bone cell adhesion, proliferation and bone tissue regeneration. It is well known that GO is hydrophilic due to the presence of oxygenated functional groups. PEEK is hydrophobic, and the water contact angle reduces to 85.52 - by blending with hydrolytic PVA. The water contact angle further reduces to 78.16 - by adding 1wt% GO to PEEK-PVA. As a result, the water absorption of PEEK-PVA/1wt% GO scaffold increases with immersion time in PBS at 37 - C, leading to the dissolution of PVA phase and weight loss increment accordingly. Some pores are formed on the scaffold surface due to the PVA degradation, and the number of pores increases with immersion time [fig_ref] Figure 3: Current strategies typically used to improve bioactivity of PEEK [/fig_ref]. The GO sheets are known to promote the adhesion, growth and differentiation of osteoblasts [bib_ref] Graphene nanomaterials: Synthesis, biocompatibility, and cytotoxicity, Liao [/bib_ref] [bib_ref] Phosphate graphene as an intrinsically osteoinductive scaffold for stem cell-driven bone regeneration, Arnold [/bib_ref] [bib_ref] Graphene oxide promotes embryonic stem cell differentiation to haematopoietic lineage, Garcia-Alegria [/bib_ref]. Therefore, PEEK-PVA/1 wt% GO scaffold promotes the attachment, proliferation and differentiation of MG63 cells as revealed by live/dead assay and CCK-8 assay results and encourages new bone formation in New Zealand white rabbits in vivo. [fig_ref] Table 3: The strengths and weaknesses of 3D-printed PEEK-based scaffolds [/fig_ref] lists the strengths and weaknesses of 3D-printed PEEK-based scaffolds prepared by additive manufacturing process. [fig_ref] Table 4: Compressive mechanical properties, in vitro and in vivo animal test results of... [/fig_ref] summarizes the compression mechanical behaviors, in vitro and in vivo animal test properties of these scaffolds. [fig_ref] Table 4: Compressive mechanical properties, in vitro and in vivo animal test results of... [/fig_ref] summarizes the compression mechanical behaviors, in vitro and in vivo animal test properties of these scaffolds. [bib_ref] Recent advances on the measurement and calculation of the elastic moduli of..., Novitskaya [/bib_ref] In general, the translation of newly developed scaffolds from the laboratories to clinical trials involves a long-term period of intense research and testing. It depends on the successful completion of a series of technical requirements and measurements including in vitro cell culture tests, biomechanical measurements for assessing BIC, in vivo animal models for evaluating the repair potential of bone scaffolds, and ethical approval for human trials. In terms of in vitro cell culture tests, several cell types such as osteoblasts, fibroblasts and hMSCs are needed for assessing cell adhesion, growth and differentiation on the scaffolds. The hMSCs cultivation is particularly useful because they can differentiate to osteoblastic lineages on the scaffolds by expressing osteogenic marker genes such as COL1, ALP, osteopontin and osteocalcin [bib_ref] Osteogenic programming of human mesenchymal stem cells with highly efficient intracellular delivery..., Thiagarajan [/bib_ref]. The development of 3D nondegradable PEEK and degradable PEEK scaffolds for bone tissue has attracted attention in materials community very recently. At present, the researchers have only cultivated murine MC3T3-E1 and human MG63 cells on those scaffolds [bib_ref] Additively-manufactured poly-ether-ether-ketone (PEEK) lattice scaffolds with uniform microporous architectures for enhanced cellular..., Su [/bib_ref] [bib_ref] Polyetheretherketone/poly (glycolic acid) blend scaffolds with biodegradable properties, Shuai [/bib_ref] [bib_ref] Characterization and bioactivity evaluation of (polyetheretherketone/polyglycolicacid)-hydroyapatite scaffolds for tissue regeneration, Shuai [/bib_ref] [bib_ref] Graphene oxide-driven interfacial coupling in laser 3D printed PEEK/PVA scaffolds for bone..., Feng [/bib_ref]. No works have been reported in the literature on the differentiation of hMSCs on those scaffolds. For in vivo animal models, the repair ability and bone formation of newly developed scaffolds in different animal species, including mouse, rabbit, goat, dog, etc., can be assessed. Therefore, proper selection of an animal model, location of bone defect for scaffold treatment, and the age and health condition of the model should be taken into consideration [bib_ref] Animal models for bone tissue engineering and modelling disease, Mcgovern [/bib_ref]. Till to present, only one rabbit model has been reported relating new bone formation in the PEEK-PVA/1%GO scaffold [bib_ref] Graphene oxide-driven interfacial coupling in laser 3D printed PEEK/PVA scaffolds for bone..., Feng [/bib_ref]. Based on these literature reports, more MSCs cell culture and in vivo animal model tests are needed to assess the repair ability, safety and efficacy of PEEK-based scaffolds prior to human trials. Thanks to the wide applications of PEEK in spinal fusion devices as mentioned above, the information relating clinical trials of PEEK spinal fusion devices can be obtained accordingly. Such information is particularly useful for reducing the clinical trial periods and unnecessary biological testing of PEEK-based scaffolds.
## Future prospects and challenges
The majority of PEEK implants for orthopedic applications are cervical and lumbar spinal cages. In particular, surface porous PEEK implants prepared by melt-extrusion and porogen leaching can generate rough surfaces for osteoblastic cell adhesion, growth and differentiation, thereby facilitating bone ingrowth. Ti-coated PEEK implants prepared by plasma spraying also exhibit rough surfaces, acting as the sites for bone cell adhesion and proliferation as well as upregulated ALP activity and BMP-2 levels [bib_ref] Osteoblasts exhibit a more differentiated phenotype and increased bone morphogenetic protein production..., Olivares-Navarrete [/bib_ref]. Those implants exhibit large pullout force as revealed by biomechanical pullout tests, resulting from mechanical interlocking of bone with the rough coating surface. The enhanced osteogenic differentiation in vitro and large pullout force in vivo lead to the implementation of Ti-PEEK implants for posterior lumbar interbody fusion (PLIF) surgery [bib_ref] Computed tomography color mapping for evaluation of bone ongrowth on the surface..., Makino [/bib_ref]. However, such implants suffer from the delamination and wear of metallic Ti layer [bib_ref] Does impaction of titanium-coated interbody fusion cages into the disc space cause..., Kienle [/bib_ref]. The delamination of Ti coating could cause implant loosening. The wear debris of Ti particles would induce an immune response and chronic inflammation. Therefore, many challenges remain in solving the wear issues of Ti-PEEK implants. Unless the wear problem is solved, one would prefer the use of surface porous PEEK implants with enhanced osseointegration than Ti-PEEK for spinal fusion surgery.
The mechanical strength of PEEK/mHA microcomposites is insufficient for load bearing applications due to debonding of mHA fillers from the PEEK matrix. The tensile strength of these microcomposites decreases with increasing mHA content. Silane coupling agents cannot be used for enhancing the filler-matrix interactions due to adverse health effects. In this respect, nHA fillers are incorporated into PEEK to form PEEK/nHA nanocomposites. From Tables 1 and 2, the stiffness of PEEK can be increased to 6.20 ± 0.13 GPa and 7.85 ± 0.11 GPa by adding 30 wt% and 40 wt% nHA, respectively. The PEEK/40 wt% nHA with a tensile elongation of 0.69 ± 0.21% is very brittle. Thus, a balance must be maintained between the stiffness and ductility in the design of biocomposites. As such, low MWNT contents (i.e., 1.5 and 3 wt%) are added to PEEK/30 wt% nHA composite to form hybrids. The PEEK/30 wt% nHA-3.0 wt% MWNT hybrid with a stiffness of 7.13 ± 0.12 GPa, tensile strength of 64.48 ± 8.51 MPa and fracture elongation of 1.74 ± 0.58% show similar tensile properties with those of cortical bone.
From the perspective of health, the potential risks from exposure to MWNTs must be assessed [bib_ref] Safe clinical use of carbon nanotubes as innovative biomaterials, Saito [/bib_ref]. Considering nanometer dimension of MWNTs, they can penetrate easily into human cells through several ways including skin penetration, inhalation, injection and ingestion. MWNTs contain catalytic metal nanoparticles (e.g., Fe, Ni, Co, etc.), which are needed for the synthesis of nanotubes [bib_ref] Methods for carbon nanotubes synthesis-Review, Prased [/bib_ref]. Those metal catalysts together with needle-like feature of nanotubes would induce ROS and oxidative stress, leading to apoptosis of several human cell types [bib_ref] Safe clinical use of carbon nanotubes as innovative biomaterials, Saito [/bib_ref] [bib_ref] Pulmonary toxicity and fibrogenic response of carbon nanotubes, Manke [/bib_ref]. For the PEEK/30 wt%nHA-3.0 wt% MWNT hybrid, the toxic effects of MWNTs are minimized because they are embedded firmly in the PEEK matrix. On the other hand, GO sheets without metal catalysts are considered as an alternative reinforcing material [bib_ref] Graphene nanomaterials: Synthesis, biocompatibility, and cytotoxicity, Liao [/bib_ref]. However, low loading levels of GOs are rather difficult to disperse homogenously in the PEEK matrix during melt compounding and injection molding. Little enhancement in stiffness is achieved in injection molded PEEK/(0.1-5 wt%)GO nanocomposites as mentioned previously [bib_ref] Super tough graphene oxide reinforced polyetheretherketone for potential hard tissue repair applications, He [/bib_ref]. More studies relating the fabrication, in vitro cell culture and in vivo animal model of injection molded PEEK/nHA-GO are needed in near future to ensure their suitably for making load-bearing implants.
It remains a great challenge for the researchers to design load-bearing hip implants. The long-term success of artificial femoral stems primarily depends on their safe integration into the host tissues. PEEK/CF and PEEK/SCF composites are particularly suitable for load-bearing hip stems due to the stiffening and strengthening effects of carbon fibers. In particular, the tensile properties of PEEK/30 wt% SCF composite resemble those of cortical bone. This composite also possesses favorable wear resistant properties on a hip simulator for 10 million cycles. The biocompatibility of PEEK/SCF composites can be improved by adding nHA [bib_ref] Polymer-based scaffolds for soft-tissue engineering, Perez-Puyana [/bib_ref]. In vitro and in vivo animal model results reveal that PEEK/SCF-nHA hybrid facilitates MG63 cell attachment, proliferation and differentiation as well as osseointegration. Till to present, Ti-6Al-4V alloy is still used primarily for fabricating load-bearing femoral stems. Ti-6Al-4V alloy contains toxic V element and shows stress-shielding effect. Thus PEEK/CF and PEEK/SCF composites with mechanical stiffness and strength close to cortical bone show a great potential for replacing Ti-6Al-4V alloy in load-bearing implants. However, clinically available PEEK/CF and PEEK/SCF composites with nHA coating/filler for femoral stems have been very slow. The in vivo data relating the PEEK/CF composites for hip stem applications are very scarce in the literature. The HA-coated PEEK/CF hip stem implant in an ovine model exhibits bone on-growth fixation and minimal stress shielding [bib_ref] Novel surface modifications of carbon fiber-reinforced polyetheretherketone hip stem in an ovine..., Nakahara [/bib_ref] [bib_ref] In vivo implant fixation of carbon fiber-reinforced PEEK hip prostheses in an..., Nakahara [/bib_ref]. So, many efforts and further studies are needed for the development of biocompatible PEEK/CF and PEEK/SCF composites for hip stem applications.
Microbial contamination on hip implants, dental and spinal fusion devices is an unresolved issue that leads to the failure of implants, infection, morbidity and mortality [bib_ref] In vitro Investigation of the effect of oral bacteria in the surface..., Sridhar [/bib_ref] [bib_ref] Infection with spinal instrumentation: Review of pathogenesis, diagnosis, prevention, and management, Kasliwal [/bib_ref] [bib_ref] Bacterial contamination of the wound during primary total hip and knee replacement, Jonsson [/bib_ref]. Little information is available in the literature relating antibacterial activity of PEEK/SCF composites and PEEK/nHA nanocomposites. With the advent of nanotechnology, novel nanoparticles with bactericidal activity have been developed for biomedical applications. Wang et al. demonstrated that PEEK/40 wt% nano-FHA composite exhibits superior resistance against S. mutans [bib_ref] Polyetheretherketone/ nano-fluorohydroxyapatite composite with antimicrobial activity and osseointegration properties, Wang [/bib_ref]. Díez-Pascual et al. reported that PEEK/ZnO nanocomposites show good bactericidal activity against S. aureus and E. coli [bib_ref] Development of nanocomposites reinforced with carboxylated poly (ether ether ketone) grafted to..., Díez-Pascual [/bib_ref] [bib_ref] Development and characterization of novel poly (ether ether ketone)/ ZnO bionanocomposites, Díez-Pascual [/bib_ref]. ZnO nanoparticles can generate ROS under UV and visible light irradiation, and even in the dark for bacterial inactivation. Furthermore, ZnO NPs can release Zn 2+ ions upon direct contact with bacteria strains, thereby disrupting their cell membranes. However, ZnO NPs may have hazardous health effects. The risks from nanomaterials arise from their unknown adverse effects and properties [bib_ref] Analysis of the exposure of organisms to the action of nanomaterials, Staroń [/bib_ref]. There are no reports relating in-vivo animal models of PEEK/ZnO nanocomposites. Further animal models are required to confirm the successful clinical implementation of those nanocomposite implants with antibacterial activity.
# Conclusions
Metallic implants made from titanium alloy, cobalt chrome molybdenum alloy and austenitic stainless steel with high elastic modulus have noted complications associated with stress shielding, corrosion and cytotoxic effects of released metallic ions. Thus, PEEK with a lower modulus than metals is considered as a material substitute for metallic implants. However, hydrophobic PEEK is bioinert, showing poor osteointegration with bone tissues after implantation. The bioactivity of PEEK can be largely improved by either surface modification, coating deposition, or mHA/nHA filler addition. The former strategy includes the creation of surface porous layer on the medical implant. By combining melt extrusion and porogen leaching process, porous PEEK surface layer with a porosity of 67.3% has been developed for use in anterior cervical discectomy and fusion (ACDF) surgery (COHERE ® ) [bib_ref] Getting PEEK to stick to bone: The development of porous PEEK for..., Torstrick [/bib_ref]. Otherwise, the deposition of HA, Ti, or TiO 2 coating layer on PEEK by means of vacuum plasma spraying, aerosol deposition, cold spraying, magnetron sputtering, etc. can also improve osseointegration performance. However, plasma spraying of HA on PEEK would lead to inhomogeneous melting of HA into tricalcium and tetracalcium phosphates, degrading the performance of HA coating accordingly. So, aerosol deposition and cold spraying are adopted for coating HA on PEEK to resolve this issue. Meanwhile, plasma spraying of Ti coating on PEEK can produce a rough surface that facilitates the adhesion and growth of osteoblasts. As such, Ti-PEEK cages have been developed for posterior lumbar interbody fusion, with 24 patients participate in a clinical trial for fusion implants [bib_ref] Computed tomography color mapping for evaluation of bone ongrowth on the surface..., Makino [/bib_ref].
The elastic modulus of PEEK (3.7-4.0 GPa) is considerably lower than that of cortical bone ranging from 7 to 30 GPa. Thus, pure PEEK is not stiff enough to sustain applied stress for load-bearing implants. The modulus of PEEK can be enhanced by adding HA fillers or carbon fibers. The HA with a modulus of 120.6 GPa can stiffen PEEK [bib_ref] Ab initio elastic properties and tensile strength of crystalline hydroxyapatite, Ching [/bib_ref]. However, it requires more than 30 wt% mHA to achieve desired tensile properties and biocompatibility [bib_ref] Evaluating the bioactivity of a hydroxyapatite-incorporated polyetheretherketone biocomposite, Ma [/bib_ref]. Due to poor interfacial bonding, large mHA particulates often debond from the PEEK matrix during tensile testing. This leads to resulting composites with low mechanical strength. In this respect, carbon fibers with a stiffness ≥ 230 GPa are used to stiffen PEEK. In particular, discontinuous carbon fibers can be readily melt-compounded with PEEK in conventional extruders and injection molders for forming composites. The stiffness of PEEK/30 wt% SCF composite reaches as high as 18.5 ± 2.3 GPa [bib_ref] Use of a novel carbon fibre composite material for the femoral stem..., Scotchford [/bib_ref] , while that of PEEK/5wt% SCF composite reaches 7.37 ± 1.22 GPa. The stiffness and tensile strength of PEEK/5 wt% SCF and PEEK/30 wt% SCF composites match closely with those of cortical bone. However, PEEK/SCF composites are bioinert, showing poor osteoblastic adhesion and proliferation. By adding 25 wt% nHA to PEEK/20 wt% SCF, the resulting hybrid composite has a stiffness of 16.5 ± 0.7 GPa with good biocompatibility [bib_ref] Polymer-based scaffolds for soft-tissue engineering, Perez-Puyana [/bib_ref]. Thus, the mechanical properties and biocompatibility of PEEK/SCF-nHA hybrids can be tailored by adjusting SCF and nHA contents.
Hydrophobic PEEK shows non-degradation behavior in aqueous solutions, thus hampering its use as a scaffolding material for soft tissue engineering. The degradation of PEEK can be improved by blending with PGA and PVA. In this respect, porous PEEK blend scaffolds prepared by AM process exhibit apparent degradation in SBF and PBS solutions due to the dissolution of hydrolytic polymers. The PEEK blend scaffolds exhibit good compressive strength to support the structure of scaffolds from collapsing during the degradation of hydrolytic polymers. Finally, the biocompatibility of porous PEEK blend scaffolds can be greatly enhanced by adding nHA or GO. Funding: This research received no external funding.
## Conflicts of interest:
The authors declare no conflict of interest. 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium YSZ Yttria-stabilized zirconia
## Abbreviations
[fig] Figure 1: Multiple stages involved in the assessment of newly developed biomaterials for bone tissue engineering. Reproduced from [2] with permission of MDPI under the conditions of Creative Commons Attribution license. [/fig]
[fig] Figure 2: The design and fabrication of polyetheretherketone patient-specific implants using CAD/CAM system for jaw reconstruction. Reproduced from[31] with permission of Springer under the terms of the Creative Commons Attribution 4.0 International license. [/fig]
[fig] Figure 3: Current strategies typically used to improve bioactivity of PEEK. Reproduced from[101] with permission of MDPI under the terms and conditions of the Creative Commons Attribution license. [/fig]
[fig] Figure 4: Schematic illustration of air plasma spray set-up. Reproduced from[106] with permission of MDPI under the terms and conditions of the Creative Commons Attribution license. [/fig]
[fig] Figure 5: Schematic illustration of typical coating formation using aerosol deposition. Reproduced from[108] with permission of MDPI under the terms and conditions of the Creative Commons Attribution license. [/fig]
[fig] Figure 6: Histologic section of nHA-coated PEEK screw implanted into the rabbit tibia for 12 weeks. The threads are filled with bone tissues, showing osteoconductive feature of nHA-coated PEEK (arrow). Reprinted from[104] with permission of MDPI under the terms and conditions of the Creative Commons Attribution license. [/fig]
[fig] Figure 7: Scanning electron microscopic images showing (a) cross-sectional and (b) top views of porous Ti layer coated on PEEK through vacuum plasma spraying. Reproduced from [113] with permission of Dove Press under the Creative Commons Attribution -Non-Commercial (unported, v3.0) license. [/fig]
[fig] Figure 8: BMP-2 protein levels secreted by MG63 cells cultivated on Ti-PEEK, PEEK, and TCP surfaces. TCP: tissue culture plastic. * denotes p < 0.05 vs PEEK; # implies p < 0.05 vs TCP. Reproduced from [113] with permission of Dove Press under the Creative Commons Attribution -Non-Commercial (unported, v3.0) license. [/fig]
[fig] Figure 9: Shear strength between the bone tissue and implant for pure PEEK, A-TiO2/PEEK and R-TiO2/PEEK at different implantation periods. *p < 0.05 compared to PEEK at 4 weeks after implantation. Reproduced from[129] with permission of Hindawi under the Creative Commons Attribution license. [/fig]
[fig] Author: Contributions: Conceptualization, S.C.T.; Writing-original draft preparation, C.L., Y.L. and S.C.T.; Writing-review and editing, C.L., Y.L. and S.C.T.; Methodology, C.L.; Y.L.; Visualization, Y.L. All authors have read and agreed to the published version of the manuscript. [/fig]
[table] Table 1: Tensile properties of pure PEEK and its microcomposites. [/table]
[table] Table 2: Tensile properties of PEEK-based nanocomposites and hybrids. [/table]
[table] Table 3: The strengths and weaknesses of 3D-printed PEEK-based scaffolds.The printed scaffolds are bioinert and nondegradable. No micropores formed on the filaments of printed scaffolds for osteoblastic adhesion. The macro-pores formed between the filaments created by printing are far too large for bone cell adhesion SHPEEK Sulfonation of FFF-printed PEEK creates micropores on the filaments for bone cell adhesion Sulfuric acid residuals can damage bone cells and reduce their viability greatly. After sulfonation, the scaffolds must be rinsed in water several times to remove the residuals until they are contamination-free. So, it is a tedious process.Degradable scaffolds due to the dissolution of PGA. SLS process creates rough surface needed for bone cell adhesion High-temperature laser beam used for sintering polymer powders would degrade their properties. Raw polymer powders trap inside the fine voids of scaffolds due to printing are difficult to remove and may induce inflammation[228].GO sheets must be firmly attached in the matrix of composite scaffold. Otherwise, stand-alone or delaminated GO may induce cytotoxicity to human cells[76] [/table]
[table] Table 4: Compressive mechanical properties, in vitro and in vivo animal test results of 3D-printed PEEK-based scaffolds. [/table]
[bib_ref] Molecular analysis of chromium and cobalt-related toxicity, Scharf [/bib_ref] |
0s and 1s in marine molecular research: a regional HPC perspective
High-performance computing (HPC) systems have become indispensable for modern marine research, providing support to an increasing number and diversity of users. Pairing with the impetus offered by high-throughput methods to key areas such as non-model organism studies, their operation continuously evolves to meet the corresponding computational challenges. Here, we present a Tier 2 (regional) HPC facility, operating for over a decade at the Institute of Marine Biology, Biotechnology, and Aquaculture of the Hellenic Centre for Marine Research in Greece. Strategic choices made in design and 2 0s and 1s in marine molecular research upgrades aimed to strike a balance between depth (the need for a few high-memory nodes) and breadth (a number of slimmer nodes), as dictated by the idiosyncrasy of the supported research. Qualitative computational requirement analysis of the latter revealed the diversity of marine fields, methods, and approaches adopted to translate data into knowledge. In addition, hardware and software architectures, usage statistics, policy, and user management aspects of the facility are presented. Drawing upon the last decade's experience from the different levels of operation of the Institute of Marine Biology, Biotechnology, and Aquaculture HPC facility, a number of lessons are presented; these have contributed to the facility's future directions in light of emerging distribution technologies (e.g., containers) and Research Infrastructure evolution. In combination with detailed knowledge of the facility usage and its upcoming upgrade, future collaborations in marine research and beyond are envisioned.BackgroundThe ubiquitous marine environments (more than 70% of the global surface [1]) mold Earth's conditions to a great extent. The interconnected abiotic [2] and biotic factors (from bacteria [2] to megafauna [3]), shape biogeochemical cycles [4] and climates [5, 6] from local to global scales. In addition, marine systems have high socio-economic value [7] as an essential source of food and by supporting renewable energy and transport, among other services [8]. The study of marine environments involves a series of disciplines (scientific fields): from Biodiversity [9] and Oceanography to (eco)systems biology [10] and from Biotechnology [11] to Aquaculture [12].To shed light on the evolutionary history of (commercially important) marine species[13], as well as on how invasive species respond and adapt to novel environments[14], the analysis of their genetic stock structure is fundamental[15]. Similarly, biodiversity assessment is essential to elucidate ecosystem functioning[16]and to identify taxa with potential for bioprospecting applications[17]. Furthermore, systems biology approaches provide both theoretical and technical backgrounds in which integrative analyses flourish[18]. However, conventional methods do not offer the information needed to explore the aforementioned scientific topics.High-throughput sequencing (HTS) and sister methods have launched a new era in many biological disciplines[19,20]. These technologies allowed access to the genetic, transcript, protein, and metabolite repertoire [21] of studied taxa or populations, and facilitated the analysis of organism-environment interactions in communities and ecosystems[22]. Whole-genome sequencing and whole-transcriptome sequencing approaches provide valuable information for the study of non-model taxa[23]. This information can be further enriched by genotyping-bysequencing approaches, such as restriction site-associated DNA sequencing[24], or by investigating gene expression dynamics through Differential Expression (DE) analyses[25]. Moving from single species to assemblages, molecular-based identification and functional profiling of communities has become available through marker (metabarcoding), genome (metagenomics), or transcriptome (metatranscriptomics) sequencing from environmental samples[26]. To a great extent, these methods address the problem of how to produce and get access to the information on different biological systems and molecules.These 0s and 1s of information (i.e., the data) come along with challenges regarding their management, analysis, and integration[27]. The computational requirements for these tasks exceed the capacity of a standard laptop/desktop by far, owing to the sheer volume of the data and to the computational complexity of the bioinformatic algorithms employed for their analysis.
For example, building the de novo genome assembly of a nonmodel Eukaryote may require algorithms of nondeterministic polynomial time complexity. This analysis can reach up to several hundreds or thousands of GB of memory (RAM) [bib_ref] The present and future of de novo wholegenome assembly, Sohn [/bib_ref]. Hence, the challenges of how to exploit all these data and how to transform data into knowledge set the present framework in biological research [bib_ref] Big data bioinformatics, Greene [/bib_ref] [bib_ref] Big data in biology: the hope and present-day challenges in it, Pal [/bib_ref].
To address these computational challenges, the use of highperformance computing (HPC) systems has become essential in life sciences and systems biology [bib_ref] Lessons learned from implementing a national infrastructure in Sweden for storage and..., Lampa [/bib_ref]. HPC is the scientific field that aims at the optimal incorporation of technology, methodology, and the application thereof to achieve "the greatest computing capability possible at any point in time and technology". Such systems range from a small number to several thousands of interconnected computers (compute nodes). According to the Partnership for Advanced Computing in Europe, the European HPC facilities are categorized as: (i) European Centres (Tier 0), (ii) national centers (Tier 1), and (iii) regional centers (Tier 2). As the Partnership for Advanced Computing in Europe highlights, "computing drives science and science drives computing" in a great range of scientific fields, from the endeavor to maintain a sustainable Earth to efforts to expand the frontiers in our understanding of the universe. On top of the heavy computational requirements, biological analyses come with a series of other practical issues that often affect the bioinformaticsoriented HPC systems.
Researchers with purely biological backgrounds often lack the coding skills or even the familiarity required for working with Command Line Interfaces. Virtual Research Environments are web-based e-service platforms that are particularly useful for researchers lacking expertise and/or computing resources [bib_ref] Virtual research environments: an overview and a research agenda, Candela [/bib_ref]. Another common issue is that most analyses include a great number of steps, with the software used in each of these having equally numerous dependencies. A lack of continuous support for tools with different dependencies, as well as frequent and non-periodical versioning of the latter, often results in broken links and further compromises the reproducibility of analyses. Widely used containerization technologiese.g., Docker [bib_ref] An introduction to Docker and analysis of its performance, Rad [/bib_ref] and Singularity [bib_ref] Singularity: scientific containers for mobility of compute, Kurtzer [/bib_ref] -ensure reproducibility of software and replication of the analysis, thus partially addressing these challenges. By encapsulating software code along with all its corresponding dependencies in such containers, software packages become reproducible in any operating system in an easy-to-download-and-install fashion, on any infrastructure.
The Institute of Marine Biology Biotechnology and Aquaculture (IMBBC) has been developing a computing hub that, in conjunction with national and European Research Infrastructures (RIs), can support state-of-the-art marine research. The regional IMBBC HPC facility allows processing of data that de-rive from the Institute's sequencing platforms and expeditions and from multiple external sources in the context of interdisciplinary studies. Here, we present insights from a thorough analysis of the research supported by the facility and some of its latest usage statistics in terms of resource requirements, computational methods, and data types; the above have contributed in shaping the facility along its lifespan.
## The imbbc hpc facility: from a single server to a tier 2 system
The IMBBC HPC facility was launched in 2009 to support computational needs over a range of scientific fields in marine biology, with a focus on non-model taxa [bib_ref] HCMR HPC bioinformatics platform facilitates the marine and aquaculture genomics research in..., Lagnel [/bib_ref]. The facility was initiated as an infrastructure of the Institute of Marine Biology and Genetics of the Hellenic Centre for Marine Research. Its development has followed the development of national RIs [fig_ref] Figure 1: Evolution of the IMBBC HPC facility during the past 12 years, with... [/fig_ref] ; also see Section A1 in Zafeiropoulos et al. [bib_ref] The IMBBC HPC facility: history and configuration, usage statistics, user management and..., Zafeiropoulos [/bib_ref]. The first nodes were used to support the analysis of data sets generated from methods such as eDNA metabarcoding and multiple omics. Since 2015, the facility also supports Virtual Research Environments, including e-services and virtual laboratories. The current configuration of the facility presented herein is named Zorba [fig_ref] Figure 1: Evolution of the IMBBC HPC facility during the past 12 years, with... [/fig_ref] , Box 4) and will be upgraded within 2021 (see Section Future Directions). Hereafter, Zorba refers to the specific system setup from 2015 and onwards, while the facility throughout its lifespan will be referred to as "IMBBC HPC".
Zorba currently consists of 328 CPU cores, 2.3 TB total memory, and 105 TB storage. Job submission takes place on the 4 available computing partitions, or queues, as explained in [fig_ref] Figure 2: Block diagram of the Zorba architecture [/fig_ref]. Zorba at its current state achieves a peak performance of 8.3 trillion double-precision floating-point operations per second, or 8.3 Tflops, as estimated by LinPack benchmarking [bib_ref] The LINPACK benchmark: past, present and future, Dongarra [/bib_ref]. On top of these, a total 7.5 TB is distributed to all servers for the storage of environment and system files. Interconnection of both the compute and login nodes takes place via an infiniband interface with a capacity of 40 Gbps, which features very high throughput and very low latency. Infiniband is also used for a switched interconnection between the servers and the 4 available file systems. A thorough technical description of Zorba is available in Section A2 of Zafeiropoulos et al. [bib_ref] The IMBBC HPC facility: history and configuration, usage statistics, user management and..., Zafeiropoulos [/bib_ref].
More than 200 software packages are currently installed and available to users at Zorba, covering the most common analysis types. These tools allow assembly, HTS data preprocessing, phylogenetic tree construction, ortholog finding, and population structure modeling, to name a few. Access to these packages is provided through Environment Modules, a broadly used means of accessing software in HPC systems [bib_ref] ELIXIR-IT HPC@CINECA: high performance computing resources for the bioinformatics community, Castrignanò [/bib_ref].
During the last 2 years, Zorba has been moving from systemdependent pipelines previously developed at IMBBC (e.g., Para MetabarCoding) towards containerization of available and new pipelines/tools. A complete metabarcoding analysis tool for various marker genes (PEMA) [bib_ref] PEMA: a flexible pipeline for environmental DNA metabarcoding analysis of the 16S/18S..., Zafeiropoulos [/bib_ref] , the chained and automated use of STACKS, software for population genetics analysis from shortlength sequences [bib_ref] Stacks: an analysis tool set for population genomics, Catchen [/bib_ref] (latest version), a set of statistical functions in R for the computation of biodiversity indices, and analyses in cases of high computational demands [bib_ref] Optimized R functions for analysis of ecological community data using the R..., Varsos [/bib_ref] , as well as a programming workflow for the automation of biodiversity historical data curation (DECO) are among the in-house developed containers. The standard container/image format used on Zorba is Singularity. Singularity images can be served by any Zorba partition; Docker images can run instantly as Singularity images.
A thorough description of the software containers developed in Zorba can be found in Section D of Zafeiropoulos et al. [bib_ref] The IMBBC HPC facility: history and configuration, usage statistics, user management and..., Zafeiropoulos [/bib_ref].
Zorba's daily functioning is ensured by a core team of 4 fulltime, experienced staff: a hardware officer, 2 system administrators, and a permanent researcher in biodiversity informatics and data science.
More than 70 users (internal and external scientists), investigators, postdoctoral researchers, technicians, and doctoral/postgraduate students have gained access to the HPC infrastructure thus far. Support is provided officially through a help desk ticketing system. An average of 31 requests/month have been received (since June 2019), with the most demanded categories being troubleshooting (38.2%) and software installation (23.8%). Since October 2017, monthly meetings among HPC users have been established to regularly discuss such issues.
Proper scheduling of the submitted jobs and fair resource sharing is a major task that needs to be confronted day to day. To address this, a specific usage policy for each of the various partitions and a scheduling software tool set have been adopted in Zorba. Policy terms are dynamically adapted to the HPC hardware architecture and to the usage statistics, with revisions being discussed between the HPC core team and users. The Simple Linux Utility for Resource Management (SLURM) open-source cluster management system orchestrates the job scheduling and allocates resources, and a booking system helps users to organize their projects and administrators to monitor the resource reservations on a mid-to long-term basis. A SLURM Database Daemon (slurmdbd) has also been installed to allow logging and recording of job usage statistics into a separate SQL database (see Section C1 in Zafeiropoulos et al. [bib_ref] The IMBBC HPC facility: history and configuration, usage statistics, user management and..., Zafeiropoulos [/bib_ref]. An extended description of user and job administrations and orchestration can be found in Section C1 of Zafeiropoulos et al. [bib_ref] The IMBBC HPC facility: history and configuration, usage statistics, user management and..., Zafeiropoulos [/bib_ref].
Training has been an integral component of the HPC facility mindset since its launch and enables knowledge sharing across MSc and PhD students and researchers within and outside the Institute. Introductory courses are organized on a regular basis, aimed at familiarizing new users with Unix environments, programming, and HPC usage policy and resource allocation (e.g., job submission in SLURM). Furthermore, the IMBBC HPC facility has served, since 2011, as an international training platform for specific types of bioinformatic analyses (see Section C2 in Zafeiropoulos et al. [bib_ref] The IMBBC HPC facility: history and configuration, usage statistics, user management and..., Zafeiropoulos [/bib_ref]. For instance, the facility has provided computational resources for workshops on Microbial Diversity , Genomics and Metagenomics, Genomics in Biodiversity, Next -Generation Sequencing technologies and informatics tools fo r studying marine biodiversity and adaptation in the long term, or Ecological Data Analysis using R. The plan is to enhance and diversify the educational component of the HPC facility by providing courses on a more permanent basis and targeting a larger audience. An extensive listing of training activities is given in Section C2 of Zafeiropoulos et al. [bib_ref] The IMBBC HPC facility: history and configuration, usage statistics, user management and..., Zafeiropoulos [/bib_ref].
## Computational breakdown of the imbbc hpc-supported research
Systematic labelling of IMBBC HPC-supported published studies (n = 47) was performed to highlight their resource requirements. Each study was manually labelled with the relevant scientific field, the data acquisition method, the computational methods, and its resource requirements; all the annotations were validated by the corresponding authors (see Section D2 in Zafeiropoulos et al. [bib_ref] The IMBBC HPC facility: history and configuration, usage statistics, user management and..., Zafeiropoulos [/bib_ref]. It should be stated that the conclusions of this overview are specific to the studies conducted at IMBBC. In comparison, studies in the Biotechnology and Agriculture fields indicate contemporary and beyond-marine orientations of research at IMBBC, respectively (see Section B2 in Zafeiropoulos et al. [bib_ref] The IMBBC HPC facility: history and configuration, usage statistics, user management and..., Zafeiropoulos [/bib_ref]. In addition, 8 methods of data acquisition (experimental or in silico) have been defined [fig_ref] Figure 3: Bar chart with the number of publications that have used IMBBC HPC... [/fig_ref]. Among these methods, whole-genome sequencing and whole-transcriptome sequencing have been widely used in multiple fields (Biotechnology, Organismal Biology, Aquaculture). Conversely, Double digest restriction-site associated sequencing (ddRADseq) has been solely employed for population genetic studies in the context of Aquaculture.
The 47 published studies employed different computational methods (sets of tasks executed on the HPC facility). These studies served different purposes, from a range of bioinformatics analyses to HPC-oriented software optimization. The computational methods were categorized in 8 classes [fig_ref] Figure 4: Red bars denote published research with high resource requirements of the various... [/fig_ref]. The resource requirements of each computational method were evaluated in terms of memory usage, computational time, and storage. Reflecting the current Zorba capacity, studies which, in any part of their analysis, exceeded 128 GB of memory or/and 48 hours of running time or/and 200 GB physical space were classified as studies with high demands (see Supplementary file imbbc hpc labelling data.xlsx in Zafeiropoulos et al. [bib_ref] The IMBBC HPC facility: history and configuration, usage statistics, user management and..., Zafeiropoulos [/bib_ref].
As shown in [fig_ref] Figure 4: Red bars denote published research with high resource requirements of the various... [/fig_ref] , the 2 most commonly used computational methods have rather different resource requirements. While DE analysis shows a notable trend for both long computational times [fig_ref] Figure 4: Red bars denote published research with high resource requirements of the various... [/fig_ref] and high memory [fig_ref] Figure 4: Red bars denote published research with high resource requirements of the various... [/fig_ref] , eDNA-based community analysis does not have high resource requirements either in computation time or memory. High memory was commonly associated with computational methods, including de novo assembly; all relevant research concerned non-model taxa and involved short-read sequencing or combinations of short-and long-read sequencing. By contrast, phylogenetic analysis studies did not involve intensive RAM use; this is largely due to the fact that software used by IMBBC users adopts parallel solutions for tree construction. Long computational times [fig_ref] Figure 4: Red bars denote published research with high resource requirements of the various... [/fig_ref] were most often observed at the functional annotation step in transcriptome analysis, DE analysis, and comparative and evolutionary omics, when this step involved BLAST queries of thousands of predicted genes against large databases, such as nr (NCBI). Finally, a common challenge emerging from all bioinformatic approaches is significant storage limitations [fig_ref] Figure 4: Red bars denote published research with high resource requirements of the various... [/fig_ref] ; this challenge was associated with the use of HTS technologies that produce large amounts of raw data, the analysis of which involves the creation of numerous intermediate files.
Overall, published studies using the IMBBC HPC facility show a degree of variance with respect to the types of tools used (depending on the user, their bioinformatic literacy, and other factors), each of which is more or less optimized with respect to HPC use. Moreover, the variance in computational needs observed within each type of computational method reflects the diversity of the studied taxonomic groups. For instance, transcriptome analysis (involving de novo assembly and functional annotation steps) was employed for the study of taxa as diverse as bacteria, sponges, fungi, fish, and goose barnacles. The complexity of each of these organisms' transcriptomes can, to a large extent, explain the differences observed in computational time, memory, and storage.
Furthermore, Zorba CPU and RAM statistics collected since 2019 displayed some overall patterns, including an average computation load per month of less than or close to 50% of its max capacity (50% of 236 kilocorehours/month) for mostof the 24 months of the logging period. Memory requirements were also heterogeneous: most (90%) of the 44,000 jobs performed in the same 24-month period required less than 10 GB of RAM, and 0.30% of the jobs required more than 128 GB of RAM (i.e., exceeding the memory capacity of the main compute nodes [batch partition]). The detailed usage statistics of Zorba are described in Section B1 and Supplementary file zorba usage statistics.xlsx of Zafeiropoulos et al. [bib_ref] The IMBBC HPC facility: history and configuration, usage statistics, user management and..., Zafeiropoulos [/bib_ref].
## Scientific impact stories
Below, some examples of research results that were made possible with the IMBBC HPC facility are described. This list of use Computational nodes are split into 4 partitions with different specs and policy terms: bigmem supporting processes requiring up to 640 GB RAM, batch handling mostly (but not exclusively) parallel-driven jobs (either in a single node or across several nodes), minibatch aiming to serve parallel jobs with reduced resource requirements, and fast partition for non-intensive jobs. All servers, except file systems, run Debian 9 (kernel 4.9.0-8-amd64). CCBY icons from the Noun Project: "nfs file document icon" by IYIKON, PK; "Earth" By mungang kim, KR; "database": By Vectorstall, PK; "switch" by Bonegolem, IT cases is by no means exhaustive, but rather an attempt to highlight different fields of research supported by the facility, along with their distinct computational features.
## Invasive species range expansion detected with edna data from autonomous reef monitoring structures
The Mediterranean biodiversity and ecosystems are experiencing profound transformations owing to Lessepsian migration, international shipping, and aquaculture, which lead to the migration of nearly 1,000 alien species [bib_ref] Invading the Mediterranean Sea: biodiversity patterns shaped by human activities, Katsanevakis [/bib_ref]. The first step towards addressing the effects of these invasions is monitoring of the introduced taxa. A powerful tool in this direction has been eDNA metabarcoding, which has enchanced detection of invasive species [bib_ref] Environmental DNA (eDNA) metabarcoding assays to detect invasive invertebrate species in the..., Klymus [/bib_ref] , often preceding macroscopic detection. One such example is the first record of the nudibranch Anteaeolidiella lurana (Ev. Marcus & Er. in Greek waters in 2020. An eDNA metabarcoding analysis allowed for detection of the species with high confidence on fouling communities developed on Autonomous Reef Monitoring Structures (ARMS). This finding, confirmed with image analysis of photographic records on a later deployment period, is an example of work conducted within the framework of the European ASSEMBLE plus programme ARMS-MBON (Marine Biodiversity Observation Net- work). PEMA software [bib_ref] PEMA: a flexible pipeline for environmental DNA metabarcoding analysis of the 16S/18S..., Zafeiropoulos [/bib_ref] was used in this study, as well as in the 30-month pilot phase of ARMS-MBON [bib_ref] A Marine Biodiversity Observation Network for Genetic Monitoring of Hard-Bottom Communities (ARMS-MBON), Obst [/bib_ref].
## Providing omics resources for large genome-size, non-model taxa
Zorba has been used for building and annotating numerous de novo genome and transcriptome assemblies of marine species, such as the gilthead sea bream Sparus aurata [bib_ref] Genomic analysis of Sparus aurata reveals the evolutionary dynamics of sexbiased genes..., Pauletto [/bib_ref] or the greater amberjack Seriola dumerili [bib_ref] Full genome survey and dynamics of gene expression in the greater amberjack..., Sarropoulou [/bib_ref]. Both genome and transcriptome assemblies of species with large genomes often exceed the maximum available memory limit, eventually affecting the strategic choices for Zorba future upgrades (see Section Future Directions). For instance, building the draft genome assembly of the seagrass Halophila stipulacea (estimated genome size 3.5 GB) using Illumina short reads has been challenging even for seemingly simple tasks, such as a kmer analysis [bib_ref] The importance of genomics for deciphering the invasion success of the seagrass..., Tsakogiannis [/bib_ref]. Taking advantage of short-and long-read sequencing technologies to construct high-quality reference genomes, the near-chromosome level genome assembly of Lagocephalus sceleratus (Gmelin, 1789) was recently completed as a case study of high ecological interest due to the species' successful invasion throughout the Eastern Mediterranean. In the context of this study, an automated containerized pipeline allowing high-quality genome assemblies from Oxford Nanopore and Illumina data was developed (SnakeCube [bib_ref] SnakeCube: containerized and automated next-generation sequencing (NGS) pipelines for genome analyses in..., Angelova [/bib_ref]. The availability of standardized pipelines offers great perspective for in-depth studies of numerous marine species of interest in aquaculture and conservation biology, including rigorous phylogenomic analyses to position each species in the tree of life (e.g., Natsidis et al. [bib_ref] Phylogenomics investigation of sparids (Teleostei: Spariformes) using highquality proteomes highlights the importance..., Natsidis [/bib_ref].
## De analysis of aquaculture fish species sheds light on critical phenotypes
Distinct, observable properties, such as morphology, development, and behavior, characterize living taxa. The correspond-ing phenotypes may be controlled by the interplay between specific genotypes and the environment. To capture an individual's genotype at a specific time point, molecular tools for transcript quantification have followed the fast development of technologies, with Expressed Sequence Tags as the first approach to be historically used, especially suited for non-model taxa [bib_ref] Profiling of infection specific mRNA transcripts of the European seabass Dicentrarchus labrax, Sarropoulou [/bib_ref]. Nowadays, the physiological state of aquaculture species is retrieved through investigation of stage-specific and immune-and stress response-specific transcriptomic profiles using RNAseq. The corresponding computational workflows involve installing various tools at Zorba and implementing a series of steps that often take days to compute. These analyses, besides detecting transcripts at a specific physiological state, have successfully identified regulatory elements, such as microRNAs. Through the construction of a regulatory network with putative target genes, microRNAs have been linked to the transcriptome expression patterns. The most recent example is the identification of mi-croRNAs and their putative target genes involved in ovary maturation [bib_ref] Non-coding RNA expression patterns of two different teleost gonad maturation stages, Papadaki [/bib_ref].
## Large-scale ecological statistics: are all taxa equal?
The nomenclature of living organisms, as well as their descriptions and their classifications under a specific nomenclature code, have been studied for more than 2 centuries. Up to now, all the species present in an ecosystem have been considered equal in terms of their contributions to diversity. However, this axiom has been tested only once before, on the United Kingdom's marine animal phyla, showing the inconsistency of the traditional Linnaean classification between different major groups [bib_ref] All animals are equal, but some animals are more equal than others, Warwick [/bib_ref]. In Arvanitidis et al., the average taxonomic distinctness index ( +) and its variation (Lambda+) were calculated on a matrix deriving from the complete World Register of Marine Species [bib_ref] A decade of the World Register of Marine Species-general insights and experiences..., Vandepitte [/bib_ref] , containing more than 250,000 described species of marine animals. It is the R-vLab web application, along with its HPC high RAM back-end components (on bigmem, see Section The IMBBC HPC Facility: From a Single Server to a Tier 2 System) that made such a calculation possible. This is the first time such a hypothesis has been tested on a global scale. Preliminary results show that the 2 biodiversity indices exhibit complementary patterns and that there is a highly significant yet non-linear relationship between the number of species within a phylum and the average distance through the taxonomic hierarchy.
## Discovery of novel enzymes for bioremediation
Polychlorinated biphenyls are complex, recalcitrant pollutants that pose a serious threat to wildlife and human health. The identification of novel enzymes that can degrade such organic pollutants is being intensively studied in the emerging field of bioremediation. In the context of the Horizon 2020 Tools And S trategies to access original bioactive compounds by Cultivating MARine invertebrates and associated symbionts (TASCMAR) pr oject, global ocean sampling provided a large biobank of fungal invertebrate symbionts and, through large-scale screening and bioreactor culturing, a marine-derived fungus able to remove a polychlorinated biphenyl compound was identified for the first time. Zorba resources and domain expertise in fungal genomics were used as a Centre for the Study and Sustainable Exploitation of Marine Biological Resources (CMBR) service for the analysis of multi-omic data for this symbiont. Following genome assembly of Cladosporium sp. TM-S3 [bib_ref] Draft genome sequence of a Cladosporium species isolated from the Mesophotic Ascidian..., Gioti [/bib_ref] , transcriptome assembly and a phylogenetic analysis revealed the full diversity of the symbiont's multicopper oxidases, enzymes commonly in- volved in oxidative degradation [bib_ref] Functional and transcriptomic investigation of laccase activity in the presence of PCB29..., Nikolaivits [/bib_ref]. Among these, 2 laccaselike proteins shown to remove up to 71% of the polychlorinated biphenyl compound are now being expressed to optimize their use as novel biocatalysts. This step would not have been possible without the annotation of the Cladosporium genome with transcriptome data; mapping of the purified enzymes' LC-MS (Liquid chromatography-mass spectrometry) spectra against the set of predicted proteins allowed for identification of their corresponding sequences.
## Lessons learned
## Depth and breadth are both required for a bioinformatics-oriented hpc
In our experience, the vast majority of the analyses run at the IMBBC HPC infrastructure are CPU-intensive. RAM-intensive jobs (>128 GB RAM, see Section Computational Breakdown of the IMBBC HPC-Supported Research) represent only ∼0.3% of the total jobs executed over the last 2 years (see Section B1 in Zafeiropoulos et al. [bib_ref] The IMBBC HPC facility: history and configuration, usage statistics, user management and..., Zafeiropoulos [/bib_ref]. Despite the difference in the frequency of executed jobs with distinct requirements, serving both types of jobs and ensuring their successful completion is equally important for addressing fundamental marine research questions (as shown in Section Computational Breakdown of the IMBBC HPC-Supported Research). The need for both HPC depth (a few high-memory nodes) and breadth (a number of slimmer nodes) has been previously reported [bib_ref] Lessons learned from implementing a national infrastructure in Sweden for storage and..., Lampa [/bib_ref]. This need reflects the idiosyncrasy of different bioinformatics analysis steps, often even within the same workflow. High-memory nodes are necessary for tasks such as de novo assembly of large genomes, while the availability of as many less powerful nodes as possible can speed up the execution of less demanding tasks and free resources for other users. Future research directions and the available budget further dictate tailoring of the HPC depth and breadth. Cloudbased services-e.g., for containerized workflows-may also facilitate this process once these become more affordable.
## Quota . . . overloaded
We observed that independently of the type of analysis, storage was an issue for all Zorba users [fig_ref] Figure 4: Red bars denote published research with high resource requirements of the various... [/fig_ref]. A high percentage of these issues relate to the raw data from HTS projects. These data are permanently stored in the home directories, occupying significant space. This, in conjunction with the fact that users delete their data with great reluctance, makes storage a major issue of daily use in Zorba. In specific cases where users' quota was exceeded uncontrollably, the Zorba team has been applying compression of raw and output data in contact with the user, but this is by no means a stable strategy. More generally, with the performance of the existing storage configuration in Zorba close to reaching its limits due to the increase in users and its concurrent use, several solutions have been adopted to resolve the issue. The most long-lasting solution has been the adoption of a per user quota system to allow storage sustainability and fairness in our allocation policy. This quota system nevertheless constitutes a limiting factor in pipeline execution, since lots of software tools produce unpredictably too many intermediate files, which not only increase storage but also cause job failures due to space restrictions. We managed the above issue by adding a scratch file system as an intermediate storage area for the runtime capacity needs. Following completion of their analysis, a user retains only the useful files and the rest are permanently removed. A storage upgrade scheduled within 2021 (see Section Future Directions) is expected to alleviate current storage challenges in Zorba. However, given the ever-increasing data production (e.g., as the result of decreasing sequencing costs and/or of rising imaging technologies), the responsible storage use approaches described here remain only partial solutions to anticipated future storage needs. Centralized (Tier 1 or higher) storage solutions represent a longer-term solution, which is in line with current views on how to handle big data generated by international research consortia in a long-lasting manner.
## Continuous intercommunication among different disciplines matters
Smooth functioning of an HPC system and exploitation of its full potential for research requires stable employment of a core team of computer scientists and engineers, in close collaboration with an extended team of researchers. At least 4 disciplines are involved in Zorba-related issues: computer scientists, engineers, biologists (in the broad sense, including ecologists, genomicists, etc.), and bioinformaticians with varying degrees of literacy in biology and informatics and various domain specializations (comparative genomics, biodiversity informatics, bacterial metagenomics, etc). The continuous communication among representatives of these 4 disciplines has sub-stantially contributed to research supported by Zorba and to the evolution of the HPC system itself over time. In our experience, an HPC system cannot function effectively and for long without full-time system administrators, nor with bioinformaticians alone. Although it has not been the case since the system's onset, investment in monthly meetings, seminars, and training events (in biology, containers, domain-specific applications, and computer science; see Section The IMBBC HPC Facility: From a Single Server to a Tier 2 System) is the only way to establish stable intercommunication among different players of an HPC system. Such proximity translates into timely and adequate systems and bioinformatics analysis support, an element that in its turn translates into successful research (see Section Computational Breakdown of the IMBBC HPC-Supported Research). It should be noted that the overall good experience in connectivity among different HPC players derives from Zorba being a Tier 2 system, with a number of active permanent users in double digits. The establishment of such inter-communication was relatively straightforward to implement with periodic meetings and the assistance of ticketing and other management solutions (see Section C1 in Zafeiropoulos et al. [bib_ref] The IMBBC HPC facility: history and configuration, usage statistics, user management and..., Zafeiropoulos [/bib_ref].
## The way forward: develop locally and share and deploy centrally
The various approaches regarding the function of an HPC system are strongly related to the different viewpoints of the academic communities towards the relatively new disciplines of bioinformatics and big data. These approaches are strongly affected by national and international decisions that affect the ability to fund supercomputer systems. There are advantages in deploying bioinformatics-oriented HPC systems in centralized (Tier 0 and Tier 1) facilities: better prices at hardware purchases, easier access to HPC-tailored facilities (for instance, in terms of the cooling system and physical space), or experienced technical personnel (see also Lampa et al. [bib_ref] Lessons learned from implementing a national infrastructure in Sweden for storage and..., Lampa [/bib_ref]. However, synergies between regional (Tier 2) and centralized HPC systems are fundamental for moving forward in supporting the diverse and demanding needs of bioinformatics. An example of such synergies concerns technical solutions (e.g., containerization) that address long-standing software sharing issues. In our experience, a workflow/pipeline can be developed by experts within the context of a specific project in a regional HPC facility. Once a production version of the pipeline is packaged, it can be distributed to centralized systems to cover a broader user audience (see Section The IMBBC HPC Facility From a Single Server to a Tier 2 System). Singularity containers have been developed to utterly suit HPC environments, mostly because they permit root access of the system in all cases. In addition, Singularity is compatible with all Docker images and can be used with Graphics Processing Units (GPUs) and Message Passing Interface (MPI) applications. This is why we chose to run containers in a Singularity format at Zorba. However, as Docker containers are widely used, especially in cloud computing (see more about cloud computing in Section Cloud Computing), workflows and services produced at IMBBC are offered in both container formats. Containers are an already established technology, used by the biggest cloud providers worldwide and increasingly by non-profit research institutes. Despite indirect costs (e.g., costs to containerize legacy software), we believe that these technologies will become the norm in the future, especially in the context of reproducibility and interoperability of bioinformatics analysis.
## Software optimizations for parallel execution
The most common ways of achieving implicit or explicit parallelization in modern multicore systems for bioinformatics, computational biology, and systems biology software tools are the software threads-provided by programming languagesand/or the OpenMP API [bib_ref] OpenMP: an industry standard API for shared-memory programming, Dagum [/bib_ref]. These types of multiprocessing make good use of the available cores on a multicore system (single node), but they are not capable of combining the available CPU cores from more than 1 node. Some other software tools use MPI to spawn processing chunks to many servers and/or cores or (even better) combine MPI with OpenMP/Threads to maximize the parallelization in hybrid models of concurrency. Such designs are now used to a great extent in some cases, such as phylogeny inference software that makes use of Monte Carlo Markov Chain samplers. However, these cases are but a small number compared to the majority of bioinformatics tasks, while their usage in other analyses is low. At the hardware level, simultaneous multithreading is not enabled in the compute nodes of the IMBBC HPC infrastructure. Since the majority of analyses running on the cluster demand dedicated cores, hardware multithreading does not perform well. In our experience, the existence of more (logical) cores in compute nodes misleads the least experienced users into using more threads than the physically available ones, which slows down their executions. In comparison, assisting servers (filesystems, login nodes, web servers) make use of hardware multithreading, since they serve numerous small tasks from different users/sources that commonly contain Input/Output (I/O) operations. GPUs provide an alternative way for parallel execution, but they are supported by a limited number of bioinformatics software tools. Nevertheless, GPUs can optimize the execution process in specific, widely used bioinformatic analyses, such as sequence alignment [bib_ref] GPU-BLAST: using graphics processors to accelerate protein sequence alignment, Vouzis [/bib_ref] [bib_ref] Graphics processing units in bioinformatics, computational biology and systems biology, Nobile [/bib_ref] , image processing in microtomography (e.g., microCT), or basecalling of Nanopore raw data.
## Cloud computing
A recent alternative to traditional HPC systems, such as that described in this review, is cloud computing. Cloud computing is the way of organizing computing resources so they are provided over the Internet ("the cloud"). This paradigm of computing requires the minimum management effort possible. Cloud computing providers exist in both commercial vendors and academic/publicly funded institutions and infrastructures (for more on cloud computing for bioinformatics, see Langmead and Nellore [bib_ref] Cloud computing for genomic data analysis and collaboration, Langmead [/bib_ref]. Computing resources can be reserved from individuals, institutions, organizations, or even scientific communities. The most widely-known commercial cloud providers are the "big 3" of cloud computing-namely, Amazon Web Services, Google Cloud Platform, and Microsoft Azure-while other cloud vendors are constantly emerging. Academic/publicly funded providers are also available: e.g., the EMBL-EBI Embassy Cloud.
Cloud computing services are being increasingly adopted in research, mainly because they offer simplicity and high availability to users with reduced or even no experience in HPC systems, through web interfaces. For this type of user, the time needed for data manipulation, software installation, and usersystem interaction is significantly reduced compared to using a local HPC facility.
Container technologies, especially Docker, along with container-management systems such as Kubernetes combined with OpenStack, have been widely used in a number cloud computing systems, in particular in the research domain.
It should be noted, however, that tool experimentation and benchmarking is more limited in cloud computing compared to local facilities and is costly, since it demands additional core hours of segmented computation. In-house HPC infrastructures can be fully configured to suit specific research area needs (storage available, fast interconnection for MPI jobs, number of CPUs versus available RAM, assisting services, etc.). Moreover, in cases where InfiniBand interconnection, a computer networking communications standard, is adopted in HPC, the performance in jobs and software that take advantage of it is substantial. Given the features and advantages of each approach (mentioned above) one could foresee the scenario of combining them to address the research community needs.
## Future directions
An upgrade of the existing hardware design of Zorba has been scheduled in 2021, funded by the CMBR research infrastructure [fig_ref] Figure 1: Evolution of the IMBBC HPC facility during the past 12 years, with... [/fig_ref]. More specifically:
i. 3 nodes of 40 CPU physical cores will be added through new partitions (120 cores in total); ii. the total RAM will be increased by 3.5 TB; iii. 100 TB of cold storage will be installed and is expected to alleviate the archiving problem at the existing homes/scratch file systems; and iv. the total usable existing storage capacity for users in home and scratch partitions will be increased by approximately 100 TB.
With this upgrade, it is expected that the total computational power of Zorba will be increased by approximately 6 TFlops, while the infrastructure will be capable of serving memoryintensive jobs requiring up to 1.5 TB of RAM, hosted on a single node. Eventually, more users will be able to concurrently load and analyze big data sets on the file systems. Over the coming 2 years, Zorba is also expected to have 2 major additions:
i. the acquisition of a number of GPU nodes to build a new partition, especially for serving software that has been ported to run on GPUs; and ii. the design of a parallel file system (Ceph or Lustre) to optimize concurrent I/O operations to speed up CPU-intensive jobs.
The expectation is that the upcoming upgrade of Zorba will further enhance collaborations with external users, since the types of bioinformatic tasks supported by the infrastructure are common to other disciplines beyond marine science, such as environmental omics research in the broad term. A nationwide survey targeting the community of researchers studying the environment and adopting the same approaches (HTS, biodiversity monitoring) has revealed that their computational and training needs are on the rise (A. Gioti et al., unpublished observations). Usage peaks and valleys were observed in Zorba (see Section B1 in Zafeiropoulos et al. [bib_ref] The IMBBC HPC facility: history and configuration, usage statistics, user management and..., Zafeiropoulos [/bib_ref] , similarly to other HTS-oriented HPC systems [bib_ref] Lessons learned from implementing a national infrastructure in Sweden for storage and..., Lampa [/bib_ref]. It is therefore feasible to share Zorba's idling time with other scientific communities. Besides, the Zorba upgrade is very timely in coming during a period where additional computational infrastructures emerge: the Cloud infrastructure Hypatia, funded by the Greek node of ELIXIR, is entering its production phase. It will constitute a national Tier 1 HPC facility, designed to host ∼50 computational nodes of different capabilities (regular servers, GPU-enabled servers, Solid-State Drive-enabled servers, etc.) and provide users the option to either create custom virtual machines for their computational services or to upload and execute workflows of containerized scientific software packages. In this context, a strategic combination of Zorba and Hypatia is expected to contribute to a strong computational basis in Greece. It is also expected that Zorba functionality will be augmented also through its connection with the Super Computing Installations of LifeWatch ERIC (European Research Infrastructure Consortium) (e.g., Picasso facility in Malaga, Spain). Building upon the lessons learned in the last 12 years, a foreseeable challenge for the facility is the enhancement of its usage monitoring to the example of international HPC systems [bib_ref] Tracking the NGS revolution: managing life science research on shared high-performance computing..., Dahlö [/bib_ref] , in order to allow even more efficient use of computational resources.
# Conclusions
Zorba is an established Tier 2 HPC regional facility operating in Crete, Greece. It serves as an interdisciplinary computing hub in the eastern Mediterranean, where studies in marine conservation, invasive species, extreme environments, and aquaculture are of great scientific and socio-economic interest. The facility has supported, since its launch over a decade ago, a number of different fields of marine research, covering all kingdoms of life; it can also share part of its resources to support research beyond the marine sciences.
The operational structure of Zorba enables continuous communication between users and administrators for more effective user support, troubleshooting, and job scheduling. More specifically, training, regular meetings, and containerization of in-house pipelines have proven constructive for all teams, students, and collaborators of IMBBC. This operational structure has evolved over the years based on the needs of the facility's users and the available resources. The practical solutions adopted-from hardware (e.g., depth/breadth balanced structure, user quotas, and temporary storage) to software (e.g., modularized bioinformatics application maintenance and containerization) and human resource management (e.g., frequent intercommunication, continuous cross-discipline training)-reflect IMBBC research to a large extent. However, and by incrementing previous reviews [bib_ref] Lessons learned from implementing a national infrastructure in Sweden for storage and..., Lampa [/bib_ref] , other Institutes and HPC facilities can be informed on the lessons learned (see Section Lessons Learned), and reflect on the computational requirement analysis of the methods presented (see Section Computational Breakdown of the IMBBC HPC-Supported Research) through the spectrum of their own research so as to plan ahead.
HPC facilities could reach a benefit greater than the sum of their capacities once they interconnect. The IMBBC HPC facility lies at the crossroad of 3 RIs, CMBR (Greek node of EMBRC-ERIC), LifeWatchGreece (Greek node of LifeWatch ERIC), and ELIXIR Greece, and via these will pursue further collaboration at larger Tier 0 and Tier 1 levels.
## Data availability
The data sets supporting the results of this article are available in the following Zenodo repository. The data pertaining to this manuscript are available the repository https://zenodo.org/recor d/4665308 under a CC BY 4.0 license. The repository is also reachable via the DOI: https://doi.org/10.5281/zenodo.4646132.
[fig] Figure 1: Evolution of the IMBBC HPC facility during the past 12 years, with hardware upgrades (blue boxes) and funding milestones (logos of RIs) highlighted. A single server that launched the bioinformatics era in 2009 evolved to the current Tier 2 system Zorba (Box 4), which allows processing of a wide variety of information from DNA sequences to biodiversity data. Different names of the facility denote distinct system architectures.The scientific fields of Aquaculture (∼40% of studies), Biodiversity (∼26% of studies), and Organismal biology (∼19% of studies) account for the majority of the research publications supported by the IMBBC HPC facility (Fig. 3; Supplementary File imbbc hpc labelling data.xlsx in Zafeiropoulos et al.[40]). [/fig]
[fig] Figure 2: Block diagram of the Zorba architecture. This is the IMBBC HPC facility architecture in its current setup, after 12 years of development. There are 2 login nodes and 1 intermediate where users may develop their analyses. [/fig]
[fig] Figure 3: Bar chart with the number of publications that have used IMBBC HPC facility resources, grouped by scientific field. The different methods for data acquisition are also presented. WGS, whole-genome sequencing; WTS, wholetranscriptome sequencing. [/fig]
[fig] Figure 4: Red bars denote published research with high resource requirements of the various computational methods employed at the IMBBC HPC facility due to (a) long computational times (>48 h), (b) high memory requirements (>128 GB), or (c) high storage requirements (>200 GB). For instance, no eDNA-based community analyses performed at Zorba thus far have required a large amounts of memory. [/fig]
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Early Changes of Mannose-Binding Lectin, H-Ficolin, and Procalcitonin in Patients with Febrile Neutropenia: A Prospective Observational Study
# Introduction
Blood stream infections (BSIs) due to invasive bacterial and fungal pathogens are major causes of infection related mortality. Gram-negative and gram-positive bacteremia account for 50%-60% of BSIs during febrile neutropenia (FN) episodes [bib_ref] New molecular and surrogate biomarker-based tests in the diagnosis of bacterial and..., Chen [/bib_ref] [bib_ref] Contribution of new diagnostic approaches to antifungal treatment plans in high-risk hematology..., Einsele [/bib_ref] [bib_ref] Epidemiology of invasive candidiasis: a persistent public health problem, Pfaller [/bib_ref]. Nonspecific signs and symptoms and conventional microbiologic methods pose some problems in the diagnosis of severe infections in neutropenic patients. Hemoculture is still the standard diagnostic method, but the positivity rate is only about 20-50% in FN episodes [bib_ref] A multicentre study to evaluate the impact of timing of caspofungin administration..., Hsu [/bib_ref] and microbial identification takes 2-6 days [bib_ref] New molecular and surrogate biomarker-based tests in the diagnosis of bacterial and..., Chen [/bib_ref]. Definition of early diagnostic markers that will guide antimicrobial treatment is critical [bib_ref] Molecular diagnosis of sepsis in neutropenic patients with hematological malignancies, Mancini [/bib_ref].
In current practice, antibacterial therapy is initiated immediately after blood cultures are obtained and before any other diagnostic procedures, in accordance with guidelines. Leukocytes and differential blood count, hemoglobin, platelets, serum glutamate oxaloacetate transaminase, serum glutamate pyruvate transaminase, lactate dehydrogenase, alkaline phosphatase, gamma glutamyltransferase, bilirubin, uric acid, creatinine, sodium, potassium, partial thromboplastin time, and C-reactive protein (CRP) are measured twice a week before and during therapy in the routine practice of our hematology section. Procalcitonin (PCT) is measured weekly throughout the neutropenic episode.
Mannose-binding lectin (MBL) is a plasma collectin (C-type lectin with a collagen-like domain) thought to have an important role in innate immunity [bib_ref] Mannan-binding lectin and its role in innate immunity, Kilpatrick [/bib_ref]. Its lectin domain recognizes sugar patterns typical of microbial surfaces, while its collagen-like region facilitates microbial uptake by phagocytic cells. MBL can activate the complement by a mechanism similar to the classical pathway, but using MBL-associated serine proteases instead of C1r and C1s. The complement system provides immediate defense against infection and has proinflammatory effects. MBL deficiency is defined as a serum level of <500 ng/mL. It is a laboratory finding that does not necessarily equate to a clinical disorder. MBL deficiency is associated with a large and heterogeneous group of disease processes. However, subnormal levels are also found in healthy people. To date, there is no consensus on the clinical relevance of MBL deficiency or its treatment.
According to the results of the largest adult cohort, MBL deficiency is not correlated with more frequent or more prolonged febrile episodes during myelosuppressive chemotherapy in adults with hematological cancer, but severe infections are more frequent in MBL-deficient patients and first severe infection develops earlier in this group compared with nondeficient patients [bib_ref] Low mannosebinding lectin concentration is associated with severe infection in patients with..., Vekemans [/bib_ref].
In this prospective study we aimed to confirm or refute these findings and to extend the investigation to one of the plasma ficolins, the Hakata antigen (H-ficolin). Ficolins share with collectins an overall quaternary structure resembling C1q and bind to bacteria and activate the complement using the lectin pathway of complement activation [bib_ref] The role of ficolins in innate immunity, Matsushita [/bib_ref]. H-ficolin might therefore be a potentially useful marker of innate immunity. In this respect, the significance of MBL and H-ficolin deficiency in FN patients and the role of consecutive CRP and PCT measurements in the etiological differentiation of fever and in establishing a follow-up protocol are investigated.
# Materials and methods
The study was planned and conducted with a prospective methodology. All patients were consecutively evaluated and included in the relevant predefined case groups. Patients hospitalized in the hematology and hematopoietic stem cell transplantation units of the Cerrahpaşa Medical School Training Hospital with any hematological malignancies and who developed at least one episode of FN between February 2011 and July 2012 were included in the study. Patients were divided into 3 diagnostic groups as "microbiologically confirmed infection", "clinically documented infection", and "fever of unknown origin" according to German guidelines. The patients were reevaluated at the end of the neutropenic episode and assigned to the relevant groups by the principle investigator, who was blinded to the laboratory results at that time.
## Study protocol
Three separate blood samples were obtained from the patients on 3 separate occasions: at baseline (between hospital admission and chemotherapy), at the onset of fever, and 72 h after the first febrile spike. Empirical antipseudomonal antimicrobial treatment (piperacillin tazobactam or cefoperazone/sulbactam for the first episode and carbapenem for recurrent episodes or in case of increased risk of extended-spectrum beta-lactamaseproducing gram-negative bacteria) was initiated in accordance with the FN guidelinesfollowing hemoculture. Some patients with prolonged neutropenia developed more than one febrile episode and data were recorded separately for each.
## Data collection
All required data were recorded on case follow-up forms from the first day of hospitalization. Demographic and clinical features including age, sex, comorbidities, vital signs, status of clinical sepsis, radiological data, microbiological data, antimicrobial treatment, and response data were recorded. Clinical and laboratory improvement within 96 h of treatment was defined as response to the antimicrobials.
## Inclusion criteria
Patients with hematologic malignancies who developed an episode of FN were included. Neutropenia was defined as an absolute neutrophil count of ≤500/mm 3 or 500-1000/mm 3 but expected to fall below ≤500/mm 3 within 24-48 h. Fever was defined as a single measurement of tympanic fever of ≥38 °C or at least 2 consecutive measurements of tympanic fever of ≥37.8 °C measured with 4 h intervals within 24 h of monitoring.
## Exclusion criteria
Patients lacking any of the 3 blood samples during follow-up were excluded, along with those under 18 years or pregnant.
## Antimicrobial treatment
There was no off-protocol intervention regarding antimicrobial use in FN episodes during the study period.
# Laboratory analysis
Blood cultures were incubated for 7 days in an automated hemoculture system (BacT ALERT 3D, bioMérieux, France). Conventional biochemical methods and automated systems (API automation pour identification, bioMérieux) were used for identification.
Antimicrobial susceptibility tests were performed using the disk diffusion method in accordance with the relevant Clinical and Laboratory Standards Institute recommendations. Statistical Analysis SPSS 16.0 was used for statistical analyses. Categorical variables were analyzed with chi-square tests and continuous variables were analyzed with Student t or Mann-Whitney U tests. The Spearman correlation test was used to evaluate correlation between continuous variables. A p-value of <0.05 was accepted as statistically significant.
# Ethical approval
This single-center, prospective, observational study was approved by the Institutional Review Board of Cerrahpaşa Medical School. All collected data were kept confidential.
# Results
A total of 82 patients were registered. Sixty-one FN episodes in 46 patients were included in the study after excluding 36 patients lacking any of the 3 serum samples or not fulfilling the inclusion criteria. Twenty-five (54%) of the patients were male and the mean age was 41.7, ranging between [bib_ref] Procalcitonin: a diagnostic marker of bacterial infection in neutropenic cancer patients with..., De Bont [/bib_ref] Eight (8/11) of the patients with microbiologically + clinically documented infection had primary bacteremia, 2 had bacteremia due to urinary tract infection, and 1 had urinary infection. Six of the pathogens isolated from blood cultures were gram-positive cocci and 4 were gram-negative bacilli. The most common gram-positive bacteria were methicillin-resistant coagulase negative staphylococci and the most common gramnegative bacterium was Escherichia coli.
Among those 17 cases with clinically documented infections, the source of infection was skin and soft tissue in 4, perianal abscess in 3, catheter exit site in 3, tooth abscess in 2, pneumonia in 1, myositis in 1, tracheostomy site in 1, surgical site in 1, and tonsillitis in 1. The rate of gram-negative bacteremia was significantly higher in cases with lower MBL levels when compared to cases with normal MBL levels (p=0.006) [fig_ref] Table 2: Rates and distribution of bacteremia according to mannose-binding lectin levels [/fig_ref].
The average level of MBL was 3.060 ng/mL. Average levels of MBL did not significantly vary between the 3 measurements (MBL-0, MBL-1, and MBL-2) during the episodes of FN (p=0.076) . Similarly, there was no significant difference between baseline, first day of fever, and third day of fever levels of H-ficolin (p>0.05) . The average H-ficolin level of the cases was measured as 18.470 ng/mL. Median baseline CRP level (CRP-0) was measured as 24 mg/L (normal range: 0-5 mg/L). The average CRP level was elevated to 84.8 mg/L on the first day of FN episodes (CRP-1) and to 98 mg/L on the third day (CRP-2) . This increase in the serial CRP levels was statistically significant (p<0.0001). Median PCT levels (normal range: <0.5 ng/mL) were also significantly elevated in FN episodes (baseline PCT-0: 2.13 ng/ mL, first day of fever PCT-1: 6.69 ng/mL, third day of fever PCT-2: 6.20 ng/mL; p<0.0001) .
As shown in [fig_ref] Table 3: Median serum procalcitonin and C-reactive protein levels in three patient groups with... [/fig_ref] , PCT-1 was increased with borderline significance (p=0.055), while PCT-2 was significantly higher (p=0.028) when compared to baseline levels in cases with microbiologically documented infection. Kruskal-Wallis variance analysis revealed no significant difference in terms of CRP levels between predefined subgroups, while median PCT levels were significantly higher in those with microbiologically documented infections. Median PCT levels according to FN subgroups are shown in detail in [fig_ref] Table 3: Median serum procalcitonin and C-reactive protein levels in three patient groups with... [/fig_ref] and . The correlations between CRP, PCT, MBL, and H-ficolin levels during FN episodes were examined with Spearman correlation analysis. A strongly positive correlation was found between PCT-2 and CRP-2 values (p=0.008, r=0.39). CRP and PCT trajectories on the third day of fever were found to be parallel to each other. Similarly, there was a significant correlation between H-ficolin-2 and CRP-2 values (p=0.026, r=0.33).
While there was no significant difference between hemoculturepositive and hemoculture-negative patients in terms of CRP levels, mean and median PCT levels were significantly higher in cases with bacteremia [fig_ref] Table 3: Median serum procalcitonin and C-reactive protein levels in three patient groups with... [/fig_ref].
Mortality occurred in 9 (19.6%) of the 46 cases during the study period, 7 of which involved refractory hematologic malignancies and 2 bacteremia due to multiple-drug resistant gram-negative strains, one of which was carbapenemase-producing.
# Discussion
Early diagnostic markers would ideally reflect the severity of the infection, help classify FN episodes as low-risk and high-risk in terms of likelihood of septic complications, and not be affected by the number of leukocytes and the course of underlying disease. CRP, as an acute phase marker and the most wellknown biochemical marker of inflammation in patients with FN, was not found to be useful in the differential diagnosis of fever of unknown origin, bacteremia, and clinically documented infections in neutropenic patients. Similar to our study, CRP was found to be of no use in differential diagnosis in other studies [bib_ref] Cytokine and acute-phase reactant levels in serum of children with cancer admitted..., Riikonen [/bib_ref] [bib_ref] Markers of bacteremia in febrile neutropenic patients with hematological malignancies: procalcitonin and..., Von Lilienfeld-Toal [/bib_ref]. False negativity that can be recognized in certain patient groups such as patients with leukemia, viral infections, systemic lupus erythematosus, progressive systemic sclerosis, dermatomyositis, ulcerative colitis, Sjögren's syndrome, and cerebral infarction is an additional drawback for CRP as an acute phase marker [bib_ref] Inflammatory markers, especially the mechanism of increased CRP, Kawai [/bib_ref].
Although the levels of serum PCT were determined to be lower in neutropenic patients when compared to those with intact immune systems, studies have shown that neutropenic patients had significantly higher PCT levels on days 0 and 2 in the case of sepsis [bib_ref] Procalcitonin in patients with and without immunosuppression and sepsis, Al-Nawas [/bib_ref]. The relationship between CRP and PCT levels during FN episodes was investigated in our study, and it was found that CRP is not a sensitive marker of early infection in neutropenic patients, while PCT would be preferred in the early diagnosis of sepsis. The rise of CRP or PCT from day 1 to day 3 in any patient group was evaluated as the expected peak serum levels related to the severity of infection rather than an ongoing uncontrolled sepsis. In our study, a slightly significant difference (p=0.055) was found between the first-day PCT levels (PCT-1) of FN episodes in bacteremic and nonbacteremic patients. Patients who had a microbiologically documented infection had significantly higher PCT levels on the third day (PCT-2) of the FN episode (p<0.05). CRP levels had no correlation with the clinical subcategories of FN episode, but higher PCT levels on the first day of fever (>0.5 ng/mL) and 72 h after the first peak of fever (with a cut point of >3-fold rise) were correlated with bacteremia, and particularly with gram-negative bacteremia. Although Svaldi et al. [bib_ref] Procalcitonin reduced sensitivity and specificity in heavily leucopenic and immunosuppressed patients, Svaldi [/bib_ref] reported that PCT levels did not significantly differ whether gram-negative or gram-positive bacteria were present when leukocyte count was <1.0x10 9 /L, PCT levels were found to be higher in bacteremic patients than nonbacteremic patients and were more rapidly decreased in nondocumented infections in the studies of Akçayand Secmeer et al. [bib_ref] Role of procalcitonin and CRP in differentiating a stable from a deteriorating..., Secmeer [/bib_ref]. Nevertheless, de Bont et al. [bib_ref] Procalcitonin: a diagnostic marker of bacterial infection in neutropenic cancer patients with..., De Bont [/bib_ref] reported similar levels of initial PCT levels at the onset of fever in bacteremic and nonbacteremic patients in a cohort of 66 patients. In the same study, cases with coagulase-negative staphylococci bacteremia were found not to have significant rises in PCT levels. Similar to our study, Fleischhack et al. [bib_ref] Procalcitonin in paediatric cancer patients: its diagnostic relevance is superior to that..., Fleischhack [/bib_ref] reported that children with FN infected with gram-negative bacteremia had higher PCT levels than did gram-positive cases [bib_ref] The role of procalcitonin in febrile neutropenic patients: review of the literature, Sakr [/bib_ref].
It was concluded in a review published by Sakr et al. [bib_ref] The role of procalcitonin in febrile neutropenic patients: review of the literature, Sakr [/bib_ref] of 30 studies that PCT levels were useful to distinguish the febrile episodes of systemic infection from noninfectious causes of fever. However, the capability to differentiate gram-negative and gram-positive bacteria in the etiology was limited.
MBL deficiency is defined as a serum level of <0.1 mg/L and was found in 5-10% of healthy adults. In a prospective study [bib_ref] The role of procalcitonin in febrile neutropenic patients: review of the literature, Sakr [/bib_ref] evaluating 255 adult patients with hematologic malignancies and neutropenia, MBL levels were measured prior to the initiation of chemotherapy and on the first day of a febrile episode. MBL deficiency (<500 ng/mL) was detected in 62 (24%) of the patients.
The incidence of severe infection was higher among MBLdeficient patients than among non-MBL-deficient patients. In our study, MBL levels did not show any significant change in the first 3 days of FN. MBL levels were within normal ranges in 32 (69.5%) patients, 5 (15.1%) of whom had bacteremia due to gram-positive cocci. MBL levels were low (<500 ng/mL) in 14 (30.5%) of patients. Patients with low levels of MBL had a significantly higher rate of gram-negative bacteremia compared to patients with normal MBL levels (p=0.006), suggesting a correlation between MBL levels and risk of gram-negative infection.
In the review by Frakking et al. [bib_ref] Mannose-binding lectin (MBL) and the risk for febrile neutropenia and infection in..., Frakking [/bib_ref] investigating the correlation of infection in pediatric oncology patients with MBL deficiency and/or severity of infection, no relationship was found between low MBL levels and presence of sepsis, bacteremia, or fungal infection in 3 of the 5 studies, while the results of the other 2 studies were to the contrary. Although there are a variety of studies with different results in the literature [bib_ref] Serum levels of mannose binding lectin and the risk of fever in..., Schlapbach [/bib_ref] [bib_ref] No strong relationship between mannan binding lectin or plasma ficolins and chemotherapy-related..., Kilpatrick [/bib_ref] [bib_ref] Low levels of mannose-binding lectin do not affect occurrence of severe infections..., Bergmann [/bib_ref] [bib_ref] Association of MBL gene polymorphisms with major bacterial infection in patients treated..., Horiuchi [/bib_ref] [bib_ref] Sepsis in acute myeloid leukaemia patients receiving high-dose chemotherapy: no impact of..., Klostergaard [/bib_ref] , Peterslund et al. [bib_ref] Association between deficiency of mannose-binding lectin and severe infections after chemotherapy, Peterslund [/bib_ref] showed a significant correlation between low levels of MBL and the development of bacteremia in adult patients with hematological malignancies.
In the study of Neth et al. [bib_ref] Deficiency of mannose-binding lectin and burden of infection in children with malignancy:..., Neth [/bib_ref] comparing 24 children with MBL levels of <1000 μg/L and 38 children with MBL levels of ≥1000 μg/mL, those with lower MBL levels developed FN episodes significantly more frequently. Schlapbach et al. [bib_ref] Serum levels of mannose binding lectin and the risk of fever in..., Schlapbach [/bib_ref] detected significantly more episodes of severe bacterial infections in patients with low MBL levels (<100 μg/mL), while those with higher MBL levels (>1000 μg/mL) had more frequent FN episodes due to microbiologically nondefined etiology. Kilpatrick et al. [bib_ref] No strong relationship between mannan binding lectin or plasma ficolins and chemotherapy-related..., Kilpatrick [/bib_ref] demonstrated that patients with MBL of ≤0.1 mg/mL had significantly more major infections than no infections within the follow-up period (p<0.05). Deficiency of MBL (≤0.1 µg/ mL) was significantly more frequent in patients with serious infections when compared to those with no infection within the follow-up period (p<0.05) [bib_ref] Mannan-binding lectin and its role in innate immunity, Kilpatrick [/bib_ref] in a cohort of 128 patients with hematological malignancies treated by chemotherapy alone or combined with bone marrow transplantation. In the study of Bergmann et al. [bib_ref] Low levels of mannose-binding lectin do not affect occurrence of severe infections..., Bergmann [/bib_ref] , no significant correlation between low MBL levels and the development of infection was detected in the FN episodes of patients with acute leukemia. Nevertheless, Horiuchi et al. [bib_ref] Association of MBL gene polymorphisms with major bacterial infection in patients treated..., Horiuchi [/bib_ref] showed that low levels of a particular MBL genotype were related to severe bacterial infection. The dramatic differences reported in the studies of Peterslund et al. [bib_ref] Association between deficiency of mannose-binding lectin and severe infections after chemotherapy, Peterslund [/bib_ref] , Neth et al. [bib_ref] Deficiency of mannose-binding lectin and burden of infection in children with malignancy:..., Neth [/bib_ref] , and Schlapbach et al. [bib_ref] Serum levels of mannose binding lectin and the risk of fever in..., Schlapbach [/bib_ref] in median MBL concentrations were virtually identical to those of the other patient categories. The studies of Klostergaard et al. [bib_ref] Sepsis in acute myeloid leukaemia patients receiving high-dose chemotherapy: no impact of..., Klostergaard [/bib_ref] , Frakking et al. [bib_ref] Mannose-binding lectin (MBL) and the risk for febrile neutropenia and infection in..., Frakking [/bib_ref] , and Schlapbach et al. [bib_ref] Serum levels of mannose binding lectin and the risk of fever in..., Schlapbach [/bib_ref] revealed that infections due to gram-positive bacteria were more commonly observed in cases with low MBL levels.
MBL is one of the factors that may influence susceptibility to infection [bib_ref] Mannan-binding lectin and its role in innate immunity, Kilpatrick [/bib_ref]. MBL variant alleles (implying low levels of circulating MBL) were found to be associated with major infections in recipients of allogeneic hemopoietic stem cell transplants [bib_ref] Mannose-binding lectin gene polymorphisms are associated with major infection following allogeneic hemopoietic..., Mullighan [/bib_ref]. Measuring the baseline MBL levels might be useful to define any predisposition to infections, particularly due to gram-negative bacteria, as a conclusion of our study. Baseline MBL levels might help categorize patients into highrisk and low-risk groups. A further study investigating how baseline MBL levels correlate with Multinational Association of Supportive Care in Cancer (MASCC) scores would be of great value. Given the easy treatment of its deficiency, baseline MBL will probably be a surrogate marker for the MASCC score, despite the current cost of the test.
H-ficolin was the other collectin investigated in our study. Seven patients had low levels of H-ficolin, 2 of whom developed gram-positive and 1 of whom developed gram-negative bacteremia. Due to the small number of patients in this group, no correlation was established between low H-ficolin levels and development of infection. In 4 of the 7 patients who had lower H-ficolin levels, MBL was also low. Two of these 4 patients had bacteremia.
In our study, MBL and H-ficolin levels did not show any significant variability in any subgroup of patients within the first 3 days of FN episodes. Different studies revealed different results owing to different patient groups, use of different chemotherapy regimens, and varying features of nosocomial causative agents within centers.
# Conclusion
Obtaining baseline MBL levels seems to be useful to predict severe infections, particularly due to gram-negative bacteria, in FN patients. Consecutive PCT levels are much more correlated with microbiologically documented infections, including bacteremia, and are preferable to CRP as a follow-up marker. No significant relation was found with baseline H-ficolin levels and risk of infection, and no significant change in serum level was detected during an emerging infection. Treatment of MBL deficiency would be a useful research topic to decrease the risk of severe infections, particularly due to gram-negative bacteria in cases with neutropenia.
[fig] Figure 2, Figure 1: Median procalcitonin levels in febrile neutropenic patient subgroups. FUO: Fever of unknown origin, PCT: Serum mannose-binding lectin (a), H-ficolin (b), C-reactive protein (c), and procalcitonin (d) levels in patients.0=at initial (between hospital admission and before chemotherapy), 1=at the onset of fever, and 2=at the 72 nd hour of fever. MBL: Mannose-binding lectin, PCT: procalcitonin, CRP: C-reactive protein. [/fig]
[table] Table 1: Hematological diagnoses of patients and number of febrile neutropenia episodes. [/table]
[table] Table 2: Rates and distribution of bacteremia according to mannose-binding lectin levels. [/table]
[table] Table 3: Median serum procalcitonin and C-reactive protein levels in three patient groups with febrile neutropenia. [/table]
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Polar Chromosomes—Challenges of a Risky Path
## Introduction-chromosome congression and alignment
The aim of the mitotic process is to segregate the genetic material packed into duplicated chromosomes equally between two daughter cells. To accomplish this, cells form a highly dynamic yet robust structure called the mitotic spindle [bib_ref] Mitotic Spindle Assembly in Animal Cells: A Fine Balancing Act, Prosser [/bib_ref] [bib_ref] Self-Organization and Forces in the Mitotic Spindle, Pavin [/bib_ref]. In the majority of higher eukaryotes, chromosomes attach to spindle microtubules (MTs) by kinetochores, large macromolecular complexes that assemble specifically on the centromere of each chromosome. The establishment of stable connections between kinetochores and MTs occurs during prometaphase, a period of mitosis defined by the nuclear envelope breakdown (NEBD) and full alignment of the chromosomes in the equatorial plane of the spindle, which defines the beginning of metaphase. During early prometaphase, the cell shape changes, the interphase array of MTs is reorganized, and MT dynamics drastically increase as MTs invade an opened nuclear space packed with chromosomes [bib_ref] From the Cytoskeleton to the Nucleus: An Integrated View on Early Spindle..., Nunes [/bib_ref]. This initiates interactions between MTs and kinetochores, resulting in the formation of the mitotic spindle and the alignment of chromosomes to the spindle equator [bib_ref] Mechanisms of Chromosome Congression during Mitosis, Maiato [/bib_ref].
The final positive outcome of prometaphase from the perspective of one chromosome is the stable attachment of sister kinetochores to MTs that emanate from opposite spindle poles and form kinetochore fibers (k-fibers), which is known as amphitelic attachment [bib_ref] Mechanisms of Chromosome Behaviour during Mitosis, Walczak [/bib_ref]. From the perspective of a cell, the final outcome is a stable attachment of all kinetochores to MTs and stable alignment of all chromosomes at the equatorial plane of the spindle that leads to the formation of the metaphase plate. Cells with normal metaphase plates are prepared to quickly initiate the movement of separated chromatids to their respective spindle poles during anaphase [bib_ref] Anaphase A: Disassembling Microtubules Move Chromosomes toward Spindle Poles, Asbury [/bib_ref]. Kinetochore interaction with MTs is monitored by the spindle assembly checkpoint (SAC), complex signaling machinery that prevents the mathematical modeling of S&C with real-life parameters revealed that the duration of mitosis would exceed the experimentally observed times by several orders of magnitude [bib_ref] Computer Simulations Predict That Chromosome Movements and Rotations Accelerate Mitotic Spindle Assembly..., Paul [/bib_ref] [bib_ref] Efficient Chromosome Capture Requires a Bias in the "search-and-Capture" Process during Mitotic-Spindle..., Wollman [/bib_ref]. Therefore, additional mechanisms that accelerate MT search and kinetochore capture have been proposed, including regulated changes in cell shape occurring during early prometaphase [bib_ref] Mitotic Rounding Alters Cell Geometry to Ensure Efficient Bipolar Spindle Formation, Lancaster [/bib_ref] , prepositioning of the main components of the spindle [bib_ref] The Spatial Arrangement of Chromosomes during Prometaphase Facilitates Spindle Assembly, Magidson [/bib_ref] , guided growth of astral MTs towards the kinetochore [bib_ref] Relative Contributions of Chromatin and Kinetochores to Mitotic Spindle Assembly, O'connell [/bib_ref] , nucleation of kinetochore MTs [bib_ref] Microtubules Assemble near Most Kinetochores during Early Prometaphase in Human Cells, Sikirzhytski [/bib_ref] , pivoting of astral MTs around the spindle pole [bib_ref] Tolić-Nørrelykke, I.M. Pivoting of Microtubules around the Spindle Pole Accelerates Kinetochore Capture, Kalinina [/bib_ref] , rotation of chromosomes [bib_ref] Computer Simulations Predict That Chromosome Movements and Rotations Accelerate Mitotic Spindle Assembly..., Paul [/bib_ref] [bib_ref] How Cells Get the Right Chromosomes, Nicklas [/bib_ref] [bib_ref] Motile Kinetochores and Polar Ejection Forces Dictate Chromosome Position on the Vertebrate..., Rieder [/bib_ref] , and kinetochore expansion before MT capture [bib_ref] Adaptive Changes in the Kinetochore Architecture Facilitate Proper Spindle Assembly, Magidson [/bib_ref] [fig_ref] Figure 2: Mechanisms of kinetochore search-and-capture and gathering on the spindle [/fig_ref] , parts 2-5) (see detailed review by [bib_ref] Role of Spatial Patterns and Kinetochore Architecture in Spindle Morphogenesis, Renda [/bib_ref].
## Figure 2.
Mechanisms of kinetochore search-and-capture and gathering on the spindle. (A) (1) Chromosome alignment involves the microtubule search for kinetochores (blue circles) by dynamic growth (dotted lines and orange arrow pointing away from the microtubule tip) and shrinkage (empty white line and orange arrow pointing to the microtubule tip). The efficiency of search-andcapture is facilitated by different additional mechanisms, including (2) biased microtubule growth towards the kinetochore via the Ran-GTP gradient (blue gradient), (3) microtubule growth from the kinetochore, (4) microtubule pivoting (orange curved arrow) around the spindle pole, and (5) kinetochore expansion before microtubule capture. Microtubule plus and minus ends are denoted by encircled + and − signs. The spindle is represented as monopolar for simplicity. (B) The successful capture event for the polar chromosome is followed by the gliding of the kinetochore laterally along the microtubules mediated by dynein walking toward the minus end of microtubule (top) or by kinetochore tracking of the depolymerizing microtubule tip (empty white line) by CENP-E. The green arrow represents the direction of dynein walking, and the grey arrow represents the direction of microtubule depolymerization. (C) Chromosome gathering on the spindle is also facilitated by the synchronous actomyosin contractility (illustrated with orange arrows) during early prometaphase, independently of microtubules.
## Oscillating at the equator-maintenance of alignment
Kinetochores that become stably aligned maintain their position within the metaphase plate by moving in an oscillatory manner around the equatorial plane [bib_ref] Attenuated Chromosome Oscillation as a Cause of Chromosomal Instability in Cancer Cells, Iemura [/bib_ref] [bib_ref] Molecular Control of Kinetochore-Microtubule Dynamics and Chromosome Oscillations, Amaro [/bib_ref]. The main mechanisms that maintain chromosome alignment in vertebrate cells include the regulation of k-fiber plus end dynamics by motors such as Kif18a/kinesin-8, sliding of bridging MTs, and the action of polar ejection forces (PEFs) [bib_ref] The Kinesin-8 Motor Kif18A Suppresses Kinetochore Movements to Control Mitotic Chromosome Alignment, Stumpff [/bib_ref] [bib_ref] Elevated Polar Ejection Forces Stabilize Kinetochore-Microtubule Attachments, Cane [/bib_ref] [bib_ref] The Distribution of Polar Ejection Forces Determines the Amplitude of Chromosome Directional..., Ke [/bib_ref] [bib_ref] Overlap Microtubules Link Sister K-Fibres and Balance the Forces on Bi-Oriented Kinetochores, Kajtez [/bib_ref] [bib_ref] Microtubule-Sliding Modules Based on Kinesins EG5 and PRC1-Dependent KIF4A Drive Human Spindle..., Vukušić [/bib_ref] , though the level of contribution of each mechanism is still unclear. The relationship between the mechanisms that drive chromosome congression to the mechanisms that maintain their alignment at 1) Chromosome alignment involves the microtubule search for kinetochores (blue circles) by dynamic growth (dotted lines and orange arrow pointing away from the microtubule tip) and shrinkage (empty white line and orange arrow pointing to the microtubule tip). The efficiency of search-andcapture is facilitated by different additional mechanisms, including (2) biased microtubule growth towards the kinetochore via the Ran-GTP gradient (blue gradient), (3) microtubule growth from the kinetochore, (4) microtubule pivoting (orange curved arrow) around the spindle pole, and (5) kinetochore expansion before microtubule capture. Microtubule plus and minus ends are denoted by encircled + and − signs. The spindle is represented as monopolar for simplicity. (B) The successful capture event for the polar chromosome is followed by the gliding of the kinetochore laterally along the microtubules mediated by dynein walking toward the minus end of microtubule (top) or by kinetochore tracking of the depolymerizing microtubule tip (empty white line) by CENP-E. The green arrow represents the direction of dynein walking, and the grey arrow represents the direction of microtubule depolymerization. (C) Chromosome gathering on the spindle is also facilitated by the synchronous actomyosin contractility (illustrated with orange arrows) during early prometaphase, independently of microtubules.
## Oscillating at the equator-maintenance of alignment
Kinetochores that become stably aligned maintain their position within the metaphase plate by moving in an oscillatory manner around the equatorial plane [bib_ref] Attenuated Chromosome Oscillation as a Cause of Chromosomal Instability in Cancer Cells, Iemura [/bib_ref] [bib_ref] Molecular Control of Kinetochore-Microtubule Dynamics and Chromosome Oscillations, Amaro [/bib_ref]. The main mechanisms that maintain chromosome alignment in vertebrate cells include the regulation of k-fiber plus end dynamics by motors such as Kif18a/kinesin-8, sliding of bridging MTs, and the action of polar ejection forces (PEFs) [bib_ref] The Kinesin-8 Motor Kif18A Suppresses Kinetochore Movements to Control Mitotic Chromosome Alignment, Stumpff [/bib_ref] [bib_ref] Elevated Polar Ejection Forces Stabilize Kinetochore-Microtubule Attachments, Cane [/bib_ref] [bib_ref] The Distribution of Polar Ejection Forces Determines the Amplitude of Chromosome Directional..., Ke [/bib_ref] [bib_ref] Overlap Microtubules Link Sister K-Fibres and Balance the Forces on Bi-Oriented Kinetochores, Kajtez [/bib_ref] [bib_ref] Microtubule-Sliding Modules Based on Kinesins EG5 and PRC1-Dependent KIF4A Drive Human Spindle..., Vukušić [/bib_ref] , though the level of contribution of each mechanism is still unclear. The relationship between the mechanisms that drive chromosome congression to the mechanisms that maintain their alignment at the equator is also unclear [bib_ref] Mechanisms of Chromosome Congression during Mitosis, Maiato [/bib_ref]. However, since current alignment models rely on the presence of k-fibers at both sister kinetochores, while congression requires neither k-fibers nor a sister kinetochore [bib_ref] Chromosomes Can Congress to the Metaphase Plate before Biorientation, Kapoor [/bib_ref] [bib_ref] Biomechanics of Chromosome Alignment at the Spindle Midplane, Risteski [/bib_ref] [bib_ref] Chromosome Behavior after Laser Microirradiation of a Single Kinetochore in Mitotic PtK2..., Mcneill [/bib_ref] [bib_ref] Chromosome Congression in the Absence of Kinetochore Fibres, Cai [/bib_ref] , the concept that these mechanisms are mechanistically distinct is prevalent in the field. Regarding the function of kinetochore motors that influence chromosome congression movement, perturbation of CENP-E and chromokinesins can influence the maintenance of alignment of chromosomes at the equator [bib_ref] CENP-E Is Essential for Reliable Bioriented Spindle Attachment, but Chromosome Alignment Can..., Mcewen [/bib_ref] [bib_ref] Human Chromokinesins Promote Chromosome Congression and Spindle Microtubule Dynamics during Mitosis, Wandke [/bib_ref] [bib_ref] Unstable Kinetochore-Microtubule Capture and Chromosomal Instability Following Deletion of CENP-E, Putkey [/bib_ref] [bib_ref] Kinetochore Kinesin CENP-E Is a Processive Bi-Directional Tracker of Dynamic Microtubule Tips, Gudimchuk [/bib_ref] , but it is not clear if this is related to the same mechanical causes as during congression.
## Biomechanical and molecular aspects of polar chromosome congression
## Getting to the spindle-movements towards and across the polar region
The first movement typical for a polar chromosome is transport to the polar region of the forming spindle [bib_ref] A Kinase Contributes to a Pole-Based Error Correction Pathway, Ye [/bib_ref] [bib_ref] Aurora B Is Enriched at Merotelic Attachment Sites, Where It Regulates MCAK, Knowlton [/bib_ref] [bib_ref] Correcting Improper Chromosome-Spindle Attachments during Cell Division, Lampson [/bib_ref] [bib_ref] Kinetochores Are Transported Poleward along a Single Astral Microtubule during Chromosome Attachment..., Rieder [/bib_ref] [bib_ref] Chromosome Motion during Attachment to the Vertebrate Spindle: Initial Saltatory-like Behavior of..., Alexander [/bib_ref] [bib_ref] Directional Instability of Kinetochore Motility during Chromosome Congression and Segregation in Mitotic..., Skibbens [/bib_ref] [bib_ref] Kinetochores Capture Astral Microtubules during Chromosome Attachment to the Mitotic Spindle: Direct..., Hayden [/bib_ref]. Two mechanisms have been proposed to drive this transport, one based on MTs and kinetochore motors and the other on actin [fig_ref] Figure 2: Mechanisms of kinetochore search-and-capture and gathering on the spindle [/fig_ref]. In the first Cells 2022, 11, 1531 5 of 27 mechanism, peripheral chromosomes are transported poleward by kinetochore-bound dynein walking along astral MTs and pulling kinetochores towards the MT minus end, which is close to the spindle pole [bib_ref] Kinetochore Dynein Is Required for Chromosome Motion and Congression Independent of the..., Yang [/bib_ref] [fig_ref] Figure 2: Mechanisms of kinetochore search-and-capture and gathering on the spindle [/fig_ref]. By studying the congression of chromosomes after depletion of dynein [bib_ref] Kinetochore Motors Drive Congression of Peripheral Polar Chromosomes by Overcoming Random Arm-Ejection..., Barisic [/bib_ref] or its kinetochore-specific adaptors ZW10 [bib_ref] Kinetochore Dynein Is Required for Chromosome Motion and Congression Independent of the..., Yang [/bib_ref] and Spindly [bib_ref] Spindly/CCDC99 Is Required for Efficient Chromosome Congression and Mitotic Checkpoint Regulation, Barisic [/bib_ref] [bib_ref] Removal of Spindly from Microtubule-Attached Kinetochores Controls Spindle Checkpoint Silencing in Human..., Gassmann [/bib_ref] , it was found that the congression of about 20% of chromosomes is drastically impaired when dynein is absent from kinetochores. The maximum velocity of initial chromosome movements is comparable to the rate at which cytoplasmic dynein moves vesicles along MTs during interphase and is in the range between 10-20 µm/min in human cells [bib_ref] Kinetochore Dynein Is Required for Chromosome Motion and Congression Independent of the..., Yang [/bib_ref] [bib_ref] Kinetochore Dynein Generates a Poleward Pulling Force to Facilitate Congression and Full..., Li [/bib_ref]. For the majority of kinetochores in human cells, this fast movement is brief, and the overall displacement is about 1 µm [bib_ref] The Spatial Arrangement of Chromosomes during Prometaphase Facilitates Spindle Assembly, Magidson [/bib_ref]. Alternatively, it has been suggested that in the absence of dynein, depolymerization of astral MTs moves kinetochores with end-on tethered CENP-E motor poleward, though at a reduced velocity compared to dynein-driven movement [bib_ref] Lateral Attachment of Kinetochores to Microtubules Is Enriched in Prometaphase Rosette and..., Itoh [/bib_ref] [fig_ref] Figure 2: Mechanisms of kinetochore search-and-capture and gathering on the spindle [/fig_ref].
Interestingly, rapid movements of chromosomes are often not directed toward the spindle pole as expected but rather to a position near the center of the nascent spindle [bib_ref] The Spatial Arrangement of Chromosomes during Prometaphase Facilitates Spindle Assembly, Magidson [/bib_ref] , route 2). Why some peripheral chromosomes move rapidly poleward while others move towards the future equatorial plane of the forming spindle remains incompletely understood. As most astral MTs have a minus-end in the centrosomes, the main MT nucleation sites of the cell [bib_ref] The Dynamics of Microtubule Minus Ends in the Human Mitotic Spindle, Lecland [/bib_ref] [bib_ref] Human Centrosome Organization and Function in Interphase and Mitosis, Vasquez-Limeta [/bib_ref] , this would imply that chromosomes that come into contact with astral MTs should show characteristic poleward movement. This is probably the case for most polar chromosomes located behind the spindle pole [bib_ref] Kinetochore Motors Drive Congression of Peripheral Polar Chromosomes by Overcoming Random Arm-Ejection..., Barisic [/bib_ref] , where astral MTs display unidirectional polarity with plus ends protruding toward the cell cortex and minus ends embedded in the centrosome [bib_ref] Mitotic Spindle Assembly in Animal Cells: A Fine Balancing Act, Prosser [/bib_ref]. However, in the central region away from the spindle, chromosomes often show surprisingly fast orthogonal movements of kinetochores by which they come close to the spindle equator [bib_ref] Non-Centrosomal Microtubules at Kinetochores Promote Rapid Chromosome Biorientation during Mitosis in Human..., Renda [/bib_ref] , whose mechanism is not completely known.
As the majority of chromosomes approach the spindle region soon after NEBD, even in conditions that diminish the activity of kinetochore dynein [bib_ref] Kinetochore Motors Drive Congression of Peripheral Polar Chromosomes by Overcoming Random Arm-Ejection..., Barisic [/bib_ref] [bib_ref] Kinetochore Dynein Is Required for Chromosome Motion and Congression Independent of the..., Yang [/bib_ref] , alternative models arose that tried to explain the characteristic synchronous spindle-directed movements of chromosomes during early prometaphase. Contractility of actin filaments on the remnants of the nuclear envelope during and after NEBD has been shown to reduce chromosome scattering during the early stages of spindle assembly [bib_ref] Myosin II-Dependent Cortical Movement Is Required for Centrosome Separation and Positioning during..., Rosenblatt [/bib_ref] [fig_ref] Figure 2: Mechanisms of kinetochore search-and-capture and gathering on the spindle [/fig_ref]. Defects in this contractility were associated with an increase in the proportion of unaligned chromosomes during pseudo-metaphase and an increase in chromosome mis-segregations during anaphase. Although the contribution of actin contractility to initial chromosome movements in somatic cells is not completely clear, this mechanism is especially relevant to gathering the scattered chromosomes to a small space of the future spindle in larger cells, such as mammalian oocytes [bib_ref] A Contractile Nuclear Actin Network Drives Chromosome Congression in Oocytes, Lénárt [/bib_ref]. Large physical barriers, similar to the actin-based one that operates in oocytes, have been observed in somatic cells, including the perinuclear actin cage in epithelial cells [bib_ref] Keratin Filaments Restrict Organelle Migration into the Forming Spindle of Newt Pneumocytes, Mandeville [/bib_ref] , and less rigid barriers often termed the 'spindle matrix' derived from the nuclear envelope in other cell types [bib_ref] A Mitotic Lamin B Matrix Induced by RanGTP Required for Spindle Assembly, Tsai [/bib_ref] [bib_ref] Requirement for Nudel and Dynein for Assembly of the Lamin B Spindle..., Ma [/bib_ref] , which can contribute to the synchronous gathering of peripheral chromosomes on the spindle.
An interesting aspect of the characteristic movements of peripheral polar chromosomes during prometaphase is their passage across the large polar region [bib_ref] The Spatial Arrangement of Chromosomes during Prometaphase Facilitates Spindle Assembly, Magidson [/bib_ref] [bib_ref] Dual Pathway Spindle Assembly Increases Both the Speed and the Fidelity of..., Kaseda [/bib_ref] [bib_ref] Centrosome-Nuclear Axis Repositioning Drives the Assembly of a Bipolar Spindle Scaffold to..., Nunes [/bib_ref]. Contrary to the poleward movement of chromosomes after NEBD, the movement across the pole cannot be explained by minus or plus end directed movement of the kinetochore along a single astral MT, either end-on or laterally attached, and would thus require mechanically different mechanisms. This movement should be directed towards the metaphase plate, as this is the general direction of congression [bib_ref] Mechanisms of Chromosome Congression during Mitosis, Maiato [/bib_ref] , and it would be interesting to explore what defines the directionality of these movements and how they depend on different types of kinetochore-MT attachments.
## Getting to the equator-congression from the spindle pole to the spindle midplane
The most studied movement of polar chromosomes during prometaphase is the process of transport of chromosomes from the spindle pole to the spindle equator, i.e., congression. The congression follows some basic principles defined by the knowledge gained through decades of studying this process. First, electron microscopy (EM) and confocal microscopy images of early prometaphase spindles revealed that kinetochores that are located between the spindle pole and the equator are in direct lateral contact with the walls of MTs, whereas end-on attachments are rarely observed [bib_ref] Microtubules Assemble near Most Kinetochores during Early Prometaphase in Human Cells, Sikirzhytski [/bib_ref] [bib_ref] The Spatial Arrangement of Chromosomes during Prometaphase Facilitates Spindle Assembly, Magidson [/bib_ref] [bib_ref] Adaptive Changes in the Kinetochore Architecture Facilitate Proper Spindle Assembly, Magidson [/bib_ref] [bib_ref] Chromosomes Can Congress to the Metaphase Plate before Biorientation, Kapoor [/bib_ref] [fig_ref] Figure 3: Main features and models of CENP-E-mediated chromosome congression to the spindle midplane [/fig_ref] , part 1). It thus became widely accepted that lateral attachments often precede biorientation and formation of amphitelic attachments [bib_ref] Chromosomes Can Congress to the Metaphase Plate before Biorientation, Kapoor [/bib_ref] [bib_ref] Chromosome Congression in the Absence of Kinetochore Fibres, Cai [/bib_ref] [bib_ref] Lateral Attachment of Kinetochores to Microtubules Is Enriched in Prometaphase Rosette and..., Itoh [/bib_ref]. Second, one of the most prominent features of congression is the gradual increase in interkinetochore distance, with and without k-fibers [bib_ref] The Spatial Arrangement of Chromosomes during Prometaphase Facilitates Spindle Assembly, Magidson [/bib_ref] [fig_ref] Figure 3: Main features and models of CENP-E-mediated chromosome congression to the spindle midplane [/fig_ref] , parts 1 and 2). Third, kinetochores change their orientation with respect to the axis of the forming spindle from random to parallel orientation, independently of the formation of end-on attachments [bib_ref] The Spatial Arrangement of Chromosomes during Prometaphase Facilitates Spindle Assembly, Magidson [/bib_ref] and without large displacement and interkinetochore stretching [bib_ref] Adaptive Changes in the Kinetochore Architecture Facilitate Proper Spindle Assembly, Magidson [/bib_ref] [fig_ref] Figure 3: Main features and models of CENP-E-mediated chromosome congression to the spindle midplane [/fig_ref] , parts 1 and 2). Lastly, chromosomes are able to congress efficiently after ablation of chromosome arms or of one kinetochore [bib_ref] Relative Contributions of Chromatin and Kinetochores to Mitotic Spindle Assembly, O'connell [/bib_ref] [bib_ref] Chromosome Fragments Possessing Only One Kinetochore Can Congress to the Spindle Equator, Khodjakov [/bib_ref] , suggesting that neither forces acting on chromosome arms nor tug-of-war between sister kinetochores are prerequisites for chromosome congression [fig_ref] Figure 3: Main features and models of CENP-E-mediated chromosome congression to the spindle midplane [/fig_ref] , parts 3 and 4).
A large amount of work revealed that The essential factor responsible for chromosome congression is the CENP-E [bib_ref] Kinetochore Motors Drive Congression of Peripheral Polar Chromosomes by Overcoming Random Arm-Ejection..., Barisic [/bib_ref] [bib_ref] CENP-E Is Essential for Reliable Bioriented Spindle Attachment, but Chromosome Alignment Can..., Mcewen [/bib_ref] [bib_ref] Centromere-Associated Protein-E Is Essential for the Mammalian Mitotic Checkpoint to Prevent Aneuploidy..., Weaver [/bib_ref] [bib_ref] Unstable Kinetochore-Microtubule Capture and Chromosomal Instability Following Deletion of CENP-E, Putkey [/bib_ref] , a plus end directed kinesin from the kinesin-7 family [bib_ref] CENP-E Is a plus End-Directed Kinetochore Motor Required for Metaphase Chromosome Alignment, Wood [/bib_ref]. CENP-E is localized to the fibrous corona, a transient structure present adjacent to the outer kinetochore layer in the absence of kinetochore end-on attachments to MTs during prometaphase [bib_ref] CENP-E Is a Putative Kinetochore Motor That Accumulates Just before Mitosis, Yen [/bib_ref] [bib_ref] CENP-E, a Novel Human Centromere-Associated Protein Required for Progression from Metaphase to..., Yen [/bib_ref] [bib_ref] The Formation, Structure, and Composition of the Mammalian Kinetochore and Kinetochore Fiber, Rieder [/bib_ref] [fig_ref] Figure 3: Main features and models of CENP-E-mediated chromosome congression to the spindle midplane [/fig_ref] , similar to dynein [bib_ref] Cytoplasmic Dynein Is Localized to Kinetochores during Mitosis, Pfarr [/bib_ref]. Currently, the dominant model for congression involves CENP-E-driven kinetochore gliding laterally alongside preformed MTs [bib_ref] Chromosomes Can Congress to the Metaphase Plate before Biorientation, Kapoor [/bib_ref] [bib_ref] Leaving No-One behind: How CENP-E Facilitates Chromosome Alignment, Craske [/bib_ref] [fig_ref] Figure 3: Main features and models of CENP-E-mediated chromosome congression to the spindle midplane [/fig_ref]. Due to the opposite walking directionalities of CENP-E and dynein, the situation when they localize on the same kinetochore could result in a tug-of-war that would prevent chromosome alignment because dynein would pull the kinetochore poleward, while CENP-E would pull the same kinetochore towards the equatorial region. However, this does not occur because of tubulin detyrosination, a posttranslational modification enriched on long-lived MTs such as k-fibers, that up-regulates CENP-E activity, making it a dominant force acting on the kinetochore in this region [bib_ref] Microtubule Detyrosination Guides Chromosomes during Mitosis, Barisic [/bib_ref] [bib_ref] The Tubulin Code: A Navigation System for Chromosomes during Mitosis, Barisic [/bib_ref] [fig_ref] Figure 3: Main features and models of CENP-E-mediated chromosome congression to the spindle midplane [/fig_ref].
Based on the gliding activity of CENP-E, it is assumed that this motor delivers peripheral polar chromosomes to the area of the spindle that is favorable for the formation of proper end-on attachments [bib_ref] Role of Spatial Patterns and Kinetochore Architecture in Spindle Morphogenesis, Renda [/bib_ref]. However, it is still not clear what would define the region where biorientation is favorable. A recent model proposed that this region is defined by the extent of antiparallel overlaps of interpolar MTs [bib_ref] Non-Centrosomal Microtubules at Kinetochores Promote Rapid Chromosome Biorientation during Mitosis in Human..., Renda [/bib_ref]. In this model, transient interactions between short MTs protruding from the kinetochore, and antiparallel MTs of the spindle, help to organize kinetochore MTs into two bundles that are oriented with the minus ends facing toward the opposite spindle poles [fig_ref] Figure 3: Main features and models of CENP-E-mediated chromosome congression to the spindle midplane [/fig_ref]. An important feature of this model is that CENP-E sorts the plus ends of MTs and gathers them at the kinetochore [bib_ref] Non-Centrosomal Microtubules at Kinetochores Promote Rapid Chromosome Biorientation during Mitosis in Human..., Renda [/bib_ref] [fig_ref] Figure 3: Main features and models of CENP-E-mediated chromosome congression to the spindle midplane [/fig_ref]. However, it is currently unclear how CENP-E function in gliding along MTs is coordinated with its function in sorting MT ends and whether one excludes the other. Because of the synchronous bi-orientation of kinetochores in human cells during prometaphase [bib_ref] Non-Centrosomal Microtubules at Kinetochores Promote Rapid Chromosome Biorientation during Mitosis in Human..., Renda [/bib_ref] , the model of kinetochore-nucleated MTs also proposes that the process of chromosome biorientation is more deterministic than is usually assumed [bib_ref] Role of Spatial Patterns and Kinetochore Architecture in Spindle Morphogenesis, Renda [/bib_ref].
This model also implies the role of the Ran-GTP gradient centered on chromatin [fig_ref] Figure 2: Mechanisms of kinetochore search-and-capture and gathering on the spindle [/fig_ref] and the chromosomal passenger complex (CPC) on the kinetochore [bib_ref] Analysis of a RanGTP-Regulated Gradient in Mitotic Somatic Cells, Kaláb [/bib_ref] [bib_ref] The Chromosomal Passenger Complex Is Required for Chromatin-Induced Microtubule Stabilization and Spindle..., Sampath [/bib_ref] , which help the nucleation of MTs from kinetochores. Still, while it was shown that the Ran-GTP gradient could attract astral MT growth [bib_ref] Relative Contributions of Chromatin and Kinetochores to Mitotic Spindle Assembly, O'connell [/bib_ref] , it is not clear to what extent the Ran-GTP gradient affects the growth of short MTs from kinetochores. The interaction of these MTs with antiparallel bundles, although an important part of the model, is not essential since biorientation is only moderately delayed after depletion of the main crosslinker of antiparallel MTs PRC1 [bib_ref] Non-Centrosomal Microtubules at Kinetochores Promote Rapid Chromosome Biorientation during Mitosis in Human..., Renda [/bib_ref]. Strong connections between antiparallel bundles and k-fibers of sister kinetochores are observed during metaphase in vertebrate cells when almost every pair of sister kinetochores is linked with a bundle of antiparallel MTs called the bridging fiber [bib_ref] Overlap Microtubules Link Sister K-Fibres and Balance the Forces on Bi-Oriented Kinetochores, Kajtez [/bib_ref] [bib_ref] PRC1-labeled Microtubule Bundles and Kinetochore Pairs Show One-to-one Association in Metaphase, Polak [/bib_ref] [bib_ref] Microtubule Sliding within the Bridging Fiber Pushes Kinetochore Fibers Apart to Segregate..., Vukušić [/bib_ref]. However, the exact coordination of antiparallel bundle formation and chromosome congression is not fully understood, but interestingly, both are regulated by CENP-E [bib_ref] Kinetochore Motors Drive Congression of Peripheral Polar Chromosomes by Overcoming Random Arm-Ejection..., Barisic [/bib_ref]. Furthermore, the mechanism by which small MTs protruding from the kinetochore would quickly grow into a fully mature k-fiber, and the role of dynein in this process is unclear [fig_ref] Figure 3: Main features and models of CENP-E-mediated chromosome congression to the spindle midplane [/fig_ref] , although MT growth from the kinetochore was observed after laser ablation in Drosophila S2 cells [bib_ref] Centrosome-Independent Mitotic Spindle Formation in Vertebrates, Khodjakov [/bib_ref] [bib_ref] Kinetochore-Driven Formation of Kinetochore Fibers Contributes to Spindle Assembly during Animal Mitosis, Maiato [/bib_ref]. Similarly, in human cells, the severed k-fibers quickly reincorporate into the spindle by a dynein-mediated mechanism that connects the disconnected k-fibers with neighboring MTs [bib_ref] Direct Kinetochore-Spindle Pole Connections Are Not Required for Chromosome Segregation, Sikirzhytski [/bib_ref]. Yet, without dynein, the formation of k-fibers is not disrupted [bib_ref] Direct Kinetochore-Spindle Pole Connections Are Not Required for Chromosome Segregation, Sikirzhytski [/bib_ref]. Amplification of short and sorted kinetochore-nucleated MTs could be aided by the augmin complex [bib_ref] Augmin Accumulation on Long-Lived Microtubules Drives Amplification and Kinetochore-Directed Growth, David [/bib_ref] that contributes to kinetochore MT growth even in the absence of pre-existing centrosomal MTs.
## Getting to the equator-congression from the spindle pole to the spindle midplane
The most studied movement of polar chromosomes during prometaphase is the process of transport of chromosomes from the spindle pole to the spindle equator, i.e., congression. The congression follows some basic principles defined by the knowledge gained through decades of studying this process. First, electron microscopy (EM) and confocal microscopy images of early prometaphase spindles revealed that kinetochores that are located between the spindle pole and the equator are in direct lateral contact with the walls of MTs, whereas end-on attachments are rarely observed [bib_ref] Microtubules Assemble near Most Kinetochores during Early Prometaphase in Human Cells, Sikirzhytski [/bib_ref] [bib_ref] The Spatial Arrangement of Chromosomes during Prometaphase Facilitates Spindle Assembly, Magidson [/bib_ref] [bib_ref] Adaptive Changes in the Kinetochore Architecture Facilitate Proper Spindle Assembly, Magidson [/bib_ref] [bib_ref] Chromosomes Can Congress to the Metaphase Plate before Biorientation, Kapoor [/bib_ref] [fig_ref] Figure 3: Main features and models of CENP-E-mediated chromosome congression to the spindle midplane [/fig_ref] , part 1). It thus became widely accepted that lateral attachments often precede biorientation and formation of amphitelic attachments [bib_ref] Chromosomes Can Congress to the Metaphase Plate before Biorientation, Kapoor [/bib_ref] [bib_ref] Chromosome Congression in the Absence of Kinetochore Fibres, Cai [/bib_ref] [bib_ref] Lateral Attachment of Kinetochores to Microtubules Is Enriched in Prometaphase Rosette and..., Itoh [/bib_ref]. Second, one of the most prominent features of congression is the gradual increase in interkinetochore distance, with and without k-fibers [bib_ref] The Spatial Arrangement of Chromosomes during Prometaphase Facilitates Spindle Assembly, Magidson [/bib_ref] [fig_ref] Figure 3: Main features and models of CENP-E-mediated chromosome congression to the spindle midplane [/fig_ref] , parts 1 and 2). Third, kinetochores change their orientation with respect to the axis of the forming spindle from random to parallel orientation, independently of the formation of end-on attachments [bib_ref] The Spatial Arrangement of Chromosomes during Prometaphase Facilitates Spindle Assembly, Magidson [/bib_ref] and without large displacement and interkinetochore stretching [bib_ref] Adaptive Changes in the Kinetochore Architecture Facilitate Proper Spindle Assembly, Magidson [/bib_ref] [fig_ref] Figure 3: Main features and models of CENP-E-mediated chromosome congression to the spindle midplane [/fig_ref] , parts 1 and 2). Lastly, chromosomes are able to congress efficiently after ablation of chromosome arms or of one kinetochore [bib_ref] Relative Contributions of Chromatin and Kinetochores to Mitotic Spindle Assembly, O'connell [/bib_ref] [bib_ref] Chromosome Fragments Possessing Only One Kinetochore Can Congress to the Spindle Equator, Khodjakov [/bib_ref] , suggesting that neither forces acting on chromosome arms nor tug-of-war between sister kinetochores are prerequisites for chromosome congression [fig_ref] Figure 3: Main features and models of CENP-E-mediated chromosome congression to the spindle midplane [/fig_ref] , parts 3 and 4). 1) Congression of polar chromosomes to the equator involves the transition of lateral to end-on attachments of kinetochores (blue circles) to microtubules, (2) although congression is achievable by lateral-only interactions. Note that the sister kinetochores gradually change orientation to be parallel to the main spindle axis, and the interkinetochore distance gradually increases 1) Congression of polar chromosomes to the equator involves the transition of lateral to end-on attachments of kinetochores (blue circles) to microtubules, (2) although congression is achievable by lateral-only interactions. Note that the sister kinetochores gradually change orientation to be parallel to the main spindle axis, and the interkinetochore distance gradually increases during congression in (1) (C) Sorting model: By walking towards the plus end, CENP-E sorts small kinetochore-nucleated microtubules in a way that their plus ends are oriented toward the kinetochore, while minus ends are connected to dynein, which facilitates their poleward-directed growth by walking along antiparallel microtubules toward their minus ends.
(D) Tethering model: As the first step in end-on conversion, CENP-E is involved in the tethering of kinetochores to lateral surfaces of microtubules, while in the second step, MCAK is involved in the depolymerization (grey dashed line) and resolving of lateral microtubule attachments to kinetochores. The orange and green arrows in (B-D) represent the direction of motor movement, and the gray arrow represents the direction of microtubule depolymerization.
An alternative model of CENP-E activity postulates that this motor is required for the tethering of kinetochores to the lateral sides of MTs [bib_ref] CENP-E Combines a Slow, Processive Motor and a Flexible Coiled Coil to..., Kim [/bib_ref] as a first step in the lateral to end-on conversion [bib_ref] Lateral to End-on Conversion of Chromosome-Microtubule Attachment Requires Kinesins Cenp-e and MCAK, Shrestha [/bib_ref] [fig_ref] Figure 3: Main features and models of CENP-E-mediated chromosome congression to the spindle midplane [/fig_ref]. CENP-E is enriched on laterally bound kinetochores [bib_ref] The Microtubule-Dependent Motor Centromere-Associated Protein E (CENP-E) Is an Integral Component of..., Yao [/bib_ref]. During this gradual process, laterally attached kinetochores rarely detach in the presence of CENP-E, while kinesin-13 MCAK is required for efficient removal of kinetochore attachments to the lateral walls of the MT [fig_ref] Figure 3: Main features and models of CENP-E-mediated chromosome congression to the spindle midplane [/fig_ref] [bib_ref] Lateral to End-on Conversion of Chromosome-Microtubule Attachment Requires Kinesins Cenp-e and MCAK, Shrestha [/bib_ref]. Since the vertebrate kinetochore is bound by 10-30 MTs [bib_ref] Kinetochore Fiber Maturation in PtK1 Cells and Its Implications for the Mechanisms..., Mcewen [/bib_ref] [bib_ref] Electron Tomography Reveals Aspects of Spindle Structure Important for Mechanical Stability at..., O'toole [/bib_ref] , both the wall-tethering and the gliding model of the CENP-E function agree that end-on conversion is likely to be a gradual process in which some MTs at first remain bound laterally to kinetochores, while some become end-on attached, known as mixed lateral end-on attachment [bib_ref] Lateral to End-on Conversion of Chromosome-Microtubule Attachment Requires Kinesins Cenp-e and MCAK, Shrestha [/bib_ref] [fig_ref] Figure 3: Main features and models of CENP-E-mediated chromosome congression to the spindle midplane [/fig_ref]. A list of potential functions of kinetochore-associated CENP-E does not end here, as CENP-E has also been demonstrated to be a processive bi-directional tracker of dynamic MT tips [bib_ref] Kinetochore Kinesin CENP-E Is a Processive Bi-Directional Tracker of Dynamic Microtubule Tips, Gudimchuk [/bib_ref] , CENP-E can regulate the SAC through its interaction with BubR1 kinase [bib_ref] Human Bubr1 Is a Mitotic Checkpoint Kinase That Monitors Cenp-E Functions at..., Chan [/bib_ref] , and both are important for stabilization of kinetochore-MT end-on attachments. In conclusion, while CENP-E function is indispensable for the congression of polar chromosomes, it is still not clear what the dominant mechanisms of CENP-E function are in human cells.
## How do polar chromosomes set their distance to the spindle pole?
In addition to the role of dynein in the initial poleward motion of kinetochores [bib_ref] Kinetochore Dynein Is Required for Chromosome Motion and Congression Independent of the..., Yang [/bib_ref] , dynein depletion was reported to increase the number of polar chromosomes with stabilized end-on MT attachments and mature k-fibers in bipolar spindles [bib_ref] Kinetochore Motors Drive Congression of Peripheral Polar Chromosomes by Overcoming Random Arm-Ejection..., Barisic [/bib_ref]. Thus, the poleward movement of peripheral chromosomes mediated by dynein is not only a fundamental part of the chromosome pathway to the equator, but it represents a mode of preventing premature stable kinetochore-MT end-on attachments in the polar region of the spindle [bib_ref] Dynein Prevents Erroneous Kinetochore-Microtubule Attachments in Mitosis, Barisic [/bib_ref] [fig_ref] Figure 4: Mechanisms that set the distance between the chromosome and the spindle pole... [/fig_ref] , as polar chromosomes would otherwise be particularly prone to erroneous syntelic attachments to the neighboring pole [bib_ref] A Kinase Contributes to a Pole-Based Error Correction Pathway, Ye [/bib_ref] [bib_ref] Kinase Cooperates with CENP-E to Promote Timely Anaphase Onset, Maia [/bib_ref]. The molecular mechanisms that are responsible for the prevention of the premature establishment of end-on attachments, and implications of those for the alignment of polar chromosomes, are still under active investigation. To date, a role in destabilizing end-on attachments in the polar region has been proposed for Aurora A kinase in human somatic cells and oocytes [bib_ref] A Kinase Contributes to a Pole-Based Error Correction Pathway, Ye [/bib_ref] [bib_ref] Spatial Regulation of Kinetochore Microtubule Attachments by Destabilization at Spindle Poles in..., Chmátal [/bib_ref] [fig_ref] Figure 4: Mechanisms that set the distance between the chromosome and the spindle pole... [/fig_ref] , with evidence for the role of Aurora B kinase in somatic cells [bib_ref] Kinetochore Motors Drive Congression of Peripheral Polar Chromosomes by Overcoming Random Arm-Ejection..., Barisic [/bib_ref] [bib_ref] Kinase Cooperates with CENP-E to Promote Timely Anaphase Onset, Maia [/bib_ref]. mation of k-fibers is not disrupted [bib_ref] Direct Kinetochore-Spindle Pole Connections Are Not Required for Chromosome Segregation, Sikirzhytski [/bib_ref]. Amplification of short and sorted kinetochorenucleated MTs could be aided by the augmin complex [bib_ref] Augmin Accumulation on Long-Lived Microtubules Drives Amplification and Kinetochore-Directed Growth, David [/bib_ref] that contributes to kinetochore MT growth even in the absence of pre-existing centrosomal MTs.
An alternative model of CENP-E activity postulates that this motor is required for the tethering of kinetochores to the lateral sides of MTs [bib_ref] CENP-E Combines a Slow, Processive Motor and a Flexible Coiled Coil to..., Kim [/bib_ref] as a first step in the lateral to end-on conversion [bib_ref] Lateral to End-on Conversion of Chromosome-Microtubule Attachment Requires Kinesins Cenp-e and MCAK, Shrestha [/bib_ref] [fig_ref] Figure 3: Main features and models of CENP-E-mediated chromosome congression to the spindle midplane [/fig_ref]. CENP-E is enriched on laterally bound kinetochores [bib_ref] The Microtubule-Dependent Motor Centromere-Associated Protein E (CENP-E) Is an Integral Component of..., Yao [/bib_ref]. During this gradual process, laterally attached kinetochores rarely detach in the presence of CENP-E, while kinesin-13 MCAK is required for efficient removal of kinetochore attachments to the lateral walls of the MT [fig_ref] Figure 3: Main features and models of CENP-E-mediated chromosome congression to the spindle midplane [/fig_ref] [bib_ref] Lateral to End-on Conversion of Chromosome-Microtubule Attachment Requires Kinesins Cenp-e and MCAK, Shrestha [/bib_ref]. Since the vertebrate kinetochore is bound by 10-30 MTs [bib_ref] Kinetochore Fiber Maturation in PtK1 Cells and Its Implications for the Mechanisms..., Mcewen [/bib_ref] [bib_ref] Electron Tomography Reveals Aspects of Spindle Structure Important for Mechanical Stability at..., O'toole [/bib_ref] , both the wall-tethering and the gliding model of the CENP-E function agree that end-on conversion is likely to be a gradual process in which some MTs at first remain bound laterally to kinetochores, while some become endon attached, known as mixed lateral end-on attachment [bib_ref] Lateral to End-on Conversion of Chromosome-Microtubule Attachment Requires Kinesins Cenp-e and MCAK, Shrestha [/bib_ref] [fig_ref] Figure 3: Main features and models of CENP-E-mediated chromosome congression to the spindle midplane [/fig_ref]. A list of potential functions of kinetochore-associated CENP-E does not end here, as CENP-E has also been demonstrated to be a processive bi-directional tracker of dynamic MT tips [bib_ref] Kinetochore Kinesin CENP-E Is a Processive Bi-Directional Tracker of Dynamic Microtubule Tips, Gudimchuk [/bib_ref] , CENP-E can regulate the SAC through its interaction with BubR1 kinase [bib_ref] Human Bubr1 Is a Mitotic Checkpoint Kinase That Monitors Cenp-E Functions at..., Chan [/bib_ref] , and both are important for stabilization of kinetochore-MT end-on attachments. In conclusion, while CENP-E function is indispensable for the congression of polar chromosomes, it is still not clear what the dominant mechanisms of CENP-E function are in human cells.
## How do polar chromosomes set their distance to the spindle pole?
In addition to the role of dynein in the initial poleward motion of kinetochores [bib_ref] Kinetochore Dynein Is Required for Chromosome Motion and Congression Independent of the..., Yang [/bib_ref] , dynein depletion was reported to increase the number of polar chromosomes with stabilized end-on MT attachments and mature k-fibers in bipolar spindles [bib_ref] Kinetochore Motors Drive Congression of Peripheral Polar Chromosomes by Overcoming Random Arm-Ejection..., Barisic [/bib_ref]. Thus, the poleward movement of peripheral chromosomes mediated by dynein is not only a fundamental part of the chromosome pathway to the equator, but it represents a mode of preventing premature stable kinetochore-MT end-on attachments in the polar region of the spindle [bib_ref] Dynein Prevents Erroneous Kinetochore-Microtubule Attachments in Mitosis, Barisic [/bib_ref] [fig_ref] Figure 4: Mechanisms that set the distance between the chromosome and the spindle pole... [/fig_ref] , as polar chromosomes would otherwise be particularly prone to erroneous syntelic attachments to the neighboring pole [bib_ref] A Kinase Contributes to a Pole-Based Error Correction Pathway, Ye [/bib_ref] [bib_ref] Kinase Cooperates with CENP-E to Promote Timely Anaphase Onset, Maia [/bib_ref]. The molecular mechanisms that are responsible for the prevention of the premature establishment of end-on attachments, and implications of those for the alignment of polar chromosomes, are still under active investigation. To date, a role in destabilizing end-on attachments in the polar region has been proposed for Aurora A kinase in human somatic cells and oocytes [bib_ref] A Kinase Contributes to a Pole-Based Error Correction Pathway, Ye [/bib_ref] [bib_ref] Spatial Regulation of Kinetochore Microtubule Attachments by Destabilization at Spindle Poles in..., Chmátal [/bib_ref] [fig_ref] Figure 4: Mechanisms that set the distance between the chromosome and the spindle pole... [/fig_ref] , with evidence for the role of Aurora B kinase in somatic cells [bib_ref] Kinetochore Motors Drive Congression of Peripheral Polar Chromosomes by Overcoming Random Arm-Ejection..., Barisic [/bib_ref] [bib_ref] Kinase Cooperates with CENP-E to Promote Timely Anaphase Onset, Maia [/bib_ref]. As kinetochore proximity to the pole could be a critical factor for the stability of attachments, it is important to understand the mechanisms that determine the distance of chromosomes to spindle poles. Chromosome-associated kinesins, termed chromokinesins, including Kid and Kif4a, have been shown to be important in adjusting the distance at which chromosomes approach the spindle pole [bib_ref] Kinetochore Motors Drive Congression of Peripheral Polar Chromosomes by Overcoming Random Arm-Ejection..., Barisic [/bib_ref] [fig_ref] Figure 4: Mechanisms that set the distance between the chromosome and the spindle pole... [/fig_ref]. Kid and Kif4a are thought to act through MT-based force that 'eject' chromosomes from poles, termed PEF, which works either through the pushing of chromosome arms by MT-polymerization [bib_ref] Studies on the Ejection Properties of Asters: Astral Microtubule Turnover Influences the..., Ault [/bib_ref] or by the activity of plus end directed chromokinesins, mainly Kid, that walks along the MT and tows chromosome arms [bib_ref] Human Chromokinesins Promote Chromosome Congression and Spindle Microtubule Dynamics during Mitosis, Wandke [/bib_ref] [bib_ref] Microtubule Movements on the Arms of Mitotic Chromosomes: Polar Ejection Forces Quantified..., Brouhard [/bib_ref] [fig_ref] Figure 4: Mechanisms that set the distance between the chromosome and the spindle pole... [/fig_ref]. Thus, PEF opposes dynein activity close to the pole [fig_ref] Figure 4: Mechanisms that set the distance between the chromosome and the spindle pole... [/fig_ref]. PEF is predicted to increase near the pole where the density of MTs is high and to scale with the size of the chromosome [bib_ref] The Distribution of Polar Ejection Forces Determines the Amplitude of Chromosome Directional..., Ke [/bib_ref]. PEF is involved in the stabilization of MT attachments in this region [bib_ref] Kinetochore Motors Drive Congression of Peripheral Polar Chromosomes by Overcoming Random Arm-Ejection..., Barisic [/bib_ref] [bib_ref] Elevated Polar Ejection Forces Stabilize Kinetochore-Microtubule Attachments, Cane [/bib_ref] [bib_ref] Dynein Prevents Erroneous Kinetochore-Microtubule Attachments in Mitosis, Barisic [/bib_ref] [bib_ref] Polar Ejection Forces Promote the Conversion from Lateral to End-on Kinetochore-Microtubule Attachments..., Drpic [/bib_ref] , which could be because it pushes chromosomes outside of the Aurora A activity gradient [bib_ref] A Kinase Contributes to a Pole-Based Error Correction Pathway, Ye [/bib_ref] [fig_ref] Figure 4: Mechanisms that set the distance between the chromosome and the spindle pole... [/fig_ref] or because it increases the tension on the kinetochore. The latter possibility is consistent with the notion that polar chromosomes require constant tension away from the pole to establish stable kinetochore-MT attachments [bib_ref] Elements of Error Correction in Mitosis: Microtubule Capture, Release, and Tension, Nicklas [/bib_ref].
However, outside the polar region, the effect of PEF is probably minor in unperturbed mitosis and increases with reduction in kinetochore-based forces, as seen in monopolar spindles [bib_ref] The Chromokinesin Kid Is Necessary for Chromosome Arm Orientation and Oscillation, but..., Levesque [/bib_ref] , when k-fiber formation is perturbed [bib_ref] Chromokinesin Kid and Kinetochore Kinesin CENP-E Differentially Support Chromosome Congression without End-on..., Iemura [/bib_ref] , or when major kinetochore motors are depleted [bib_ref] Kinetochore Motors Drive Congression of Peripheral Polar Chromosomes by Overcoming Random Arm-Ejection..., Barisic [/bib_ref]. Furthermore, polar chromosome fragments in human cells after laser ablation move in random directions [bib_ref] Kinetochore Motors Drive Congression of Peripheral Polar Chromosomes by Overcoming Random Arm-Ejection..., Barisic [/bib_ref] , implying that PEF is not critical for congression exclusively toward the spindle equator. Similar conclusions were drawn from experiments including laser irradiation of a kinetochore [bib_ref] Chromosome Behavior after Laser Microirradiation of a Single Kinetochore in Mitotic PtK2..., Mcneill [/bib_ref] and mitosis with un-replicated genomes (MUGs) [bib_ref] Relative Contributions of Chromatin and Kinetochores to Mitotic Spindle Assembly, O'connell [/bib_ref] , in which kinetochores are required and sufficient for chromosome congression. Collectively, these experiments imply that the forces generated by kinetochore motors and kinetochore-MT attachments are dominant over PEFs for chromosome congression in unperturbed spindles, but the role of PEF could be very important close to the polar region.
The tug-of-war between different mechanisms that set the distance to the pole was nicely recapitulated in experiments in which the balance between different factors was changed in monopolar spindles [bib_ref] Kinetochore Motors Drive Congression of Peripheral Polar Chromosomes by Overcoming Random Arm-Ejection..., Barisic [/bib_ref] [bib_ref] Chromokinesin Kid and Kinetochore Kinesin CENP-E Differentially Support Chromosome Congression without End-on..., Iemura [/bib_ref] [fig_ref] Figure 4: Mechanisms that set the distance between the chromosome and the spindle pole... [/fig_ref]. Monopolar spindles are often used as a model to study the behavior of polar chromosomes, but one should be cautious with interpretations because monopolar spindles are characterized by end-on attachments such as syntelic and monotelic [bib_ref] Probing Spindle Assembly Mechanisms with Monastrol, a Small Molecule Inhibitor of the..., Kapoor [/bib_ref] , which are rare in unperturbed bipolar prometaphase spindles [bib_ref] The Spatial Arrangement of Chromosomes during Prometaphase Facilitates Spindle Assembly, Magidson [/bib_ref]. For example, in otherwise unperturbed monopolar spindles, chromosomes localize closer to the pole if CENP-E or chromokinesin activity is perturbed and farther away if dynein is depleted [bib_ref] Kinetochore Motors Drive Congression of Peripheral Polar Chromosomes by Overcoming Random Arm-Ejection..., Barisic [/bib_ref] [fig_ref] Figure 4: Mechanisms that set the distance between the chromosome and the spindle pole... [/fig_ref]. CENP-E in this condition probably mediates the motion of the leading kinetochore [bib_ref] Mechanisms of Chromosome Congression during Mitosis, Maiato [/bib_ref]. On the other hand, if NDC80 is perturbed in monopolar spindles, lateral attachments become dominant [bib_ref] HNuf2 Inhibition Blocks Stable Kinetochore-Microtubule Attachment and Induces Mitotic Cell Death in..., Deluca [/bib_ref] [bib_ref] Kinetochore Microtubule Dynamics and Attachment Stability Are Regulated by Hec1, Deluca [/bib_ref] , and in this case, perturbation of CENP-E activity increases the distances of kinetochore to poles, while it decreases after additional perturbation of chromokinesin Kid, similarly to bipolar spindles without k-fibers [bib_ref] Chromokinesin Kid and Kinetochore Kinesin CENP-E Differentially Support Chromosome Congression without End-on..., Iemura [/bib_ref] [fig_ref] Figure 4: Mechanisms that set the distance between the chromosome and the spindle pole... [/fig_ref]. These results imply that CENP-E plus end directed motor activity is dominant in conditions where stable MTs are present, such as during late prometaphase, while during early prometaphase, CENP-E could suppress chromosome congression by causing kinetochores to track short and unstable MTs [bib_ref] Kinetochore Kinesin CENP-E Is a Processive Bi-Directional Tracker of Dynamic Microtubule Tips, Gudimchuk [/bib_ref] [bib_ref] Chromokinesin Kid and Kinetochore Kinesin CENP-E Differentially Support Chromosome Congression without End-on..., Iemura [/bib_ref] [fig_ref] Figure 4: Mechanisms that set the distance between the chromosome and the spindle pole... [/fig_ref]. Stable MTs are also more detyrosinated, which favors CENP-E activity over dynein [bib_ref] Microtubule Detyrosination Guides Chromosomes during Mitosis, Barisic [/bib_ref] [fig_ref] Figure 4: Mechanisms that set the distance between the chromosome and the spindle pole... [/fig_ref]. Taken together, the distance of chromosomes to the spindle poles is an important factor that determines the stability of end-on attachments, probably due to the action of Aurora kinases, while the actual distance is determined by the tug-of-war between dominant kinetochore motors and the supportive action of PEFs close to the poles.
## Significance of centrosome prepositioning during prophase
The position of centrosomes prior to NEBD has been recognized as an important factor that influences mitotic fidelity [bib_ref] Role of Spatial Patterns and Kinetochore Architecture in Spindle Morphogenesis, Renda [/bib_ref]. Centrosome separation occurs in coordination with NEBD [bib_ref] Centrosome-Nuclear Axis Repositioning Drives the Assembly of a Bipolar Spindle Scaffold to..., Nunes [/bib_ref] , by Eg5/kinesin-5 driven sliding of antiparallel MTs [bib_ref] Probing Spindle Assembly Mechanisms with Monastrol, a Small Molecule Inhibitor of the..., Kapoor [/bib_ref] [bib_ref] The Bipolar Mitotic Kinesin Eg5 Moves on Both Microtubules That It Crosslinks, Kapitein [/bib_ref] , with the help of additional players, including myosin II [bib_ref] Myosin II-Dependent Cortical Movement Is Required for Centrosome Separation and Positioning during..., Rosenblatt [/bib_ref] , actin [bib_ref] Cortical Actin Dynamics Facilitate Early-Stage Centrosome Separation, Cao [/bib_ref] , and nuclear and cortical dynein [bib_ref] Nuclear Envelope-Associated Dynein Drives Prophase Centrosome Separation and Enables Eg5-Independent Bipolar Spindle..., Raaijmakers [/bib_ref] [bib_ref] Dynein Transmits Polarized Actomyosin Cortical Flows to Promote Centrosome Separation, De Simone [/bib_ref]. In non-transformed cells, centrosomes fully separate before NEBD, which is termed the prophase pathway [fig_ref] Figure 5: Models by which the degree of centrosome separation upon nuclear envelope breakdown [/fig_ref] , whereas, in tumor cell lines, centrosomes often separate after NEBD, also known as the prometaphase pathway [bib_ref] The Spatial Arrangement of Chromosomes during Prometaphase Facilitates Spindle Assembly, Magidson [/bib_ref] [bib_ref] Dual Pathway Spindle Assembly Increases Both the Speed and the Fidelity of..., Kaseda [/bib_ref] [bib_ref] Kinetochore-Generated Pushing Forces Separate Centrosomes during Bipolar Spindle Assembly, Toso [/bib_ref] [fig_ref] Figure 5: Models by which the degree of centrosome separation upon nuclear envelope breakdown [/fig_ref]. Additionally, the internal signals provided by the nucleus and the cytoskeleton predominantly position the separated centrosomes on the shortest nuclear axis before NEBD [bib_ref] The Spatial Arrangement of Chromosomes during Prometaphase Facilitates Spindle Assembly, Magidson [/bib_ref] [bib_ref] Centrosome-Nuclear Axis Repositioning Drives the Assembly of a Bipolar Spindle Scaffold to..., Nunes [/bib_ref] [bib_ref] Cytoplasmic Dynein Is Required for the Nuclear Attachment and Migration of Centrosomes..., Robinson [/bib_ref]. Several papers revealed that the degree of centrosome separation upon NEBD plays an important role in determining the types of kinetochore-MT attachments that form in early prometaphase, with a larger number of erroneous attachments and missegregations in the case of delayed separation of centrosomes at NEBD, and the formation of a prometaphase rosette [bib_ref] Dual Pathway Spindle Assembly Increases Both the Speed and the Fidelity of..., Kaseda [/bib_ref] [bib_ref] Timing of Centrosome Separation Is Important for Accurate Chromosome Segregation, Silkworth [/bib_ref] [bib_ref] Centrosome Dynamics as a Source of Chromosomal Instability, Nam [/bib_ref] [fig_ref] Figure 5: Models by which the degree of centrosome separation upon nuclear envelope breakdown [/fig_ref]. It was proposed that delayed centrosome separation promotes syntelic attachment in the vicinity of one pole, which can develop into merotelic attachment by an additional MT approach from the other pole [bib_ref] Timing of Centrosome Separation Is Important for Accurate Chromosome Segregation, Silkworth [/bib_ref] [fig_ref] Figure 5: Models by which the degree of centrosome separation upon nuclear envelope breakdown [/fig_ref] , part 1). Similar results have been reported after bipolarization of a monopolar spindle [bib_ref] Correcting Improper Chromosome-Spindle Attachments during Cell Division, Lampson [/bib_ref]. Furthermore, the geometry of the spindle during metaphase is often asymmetric after delayed or accelerated centrosome separation, which could also lead to the formation of merotelic attachments due to the altered angle of the MT approach from a more distal centrosome [bib_ref] Centrosome Dynamics as a Source of Chromosomal Instability, Nam [/bib_ref] [bib_ref] Cyclin B2 and P53 Control Proper Timing of Centrosome Separation, Nam [/bib_ref] [bib_ref] USP44 Regulates Centrosome Positioning to Prevent Aneuploidy and Suppress Tumorigenesis, Zhang [/bib_ref] [fig_ref] Figure 5: Models by which the degree of centrosome separation upon nuclear envelope breakdown [/fig_ref] , part 2). Merotelic attachments are particularly error-prone, as they can bypass error correction and SAC mechanisms [bib_ref] Merotelic Kinetochore Attachment: Causes and Effects, Gregan [/bib_ref] [bib_ref] Aurora B Is Enriched at Merotelic Attachment Sites, Where It Regulates MCAK, Knowlton [/bib_ref] [bib_ref] Correcting Improper Chromosome-Spindle Attachments during Cell Division, Lampson [/bib_ref]. A broadly similar role of centrosome separation has been reported in coalescing multipolar spindles [bib_ref] A Mechanism Linking Extra Centrosomes to Chromosomal Instability, Ganem [/bib_ref]. Additionally, pre-NEBD centrosome separation places most chromosomes between the two separated spindle poles, where alignment to the metaphase plate is rapid [bib_ref] Role of Spatial Patterns and Kinetochore Architecture in Spindle Morphogenesis, Renda [/bib_ref]. Thus, incompletely separated centrosomes increase the proportion of peripheral polar chromosomes at NEBD, consequently placing more chromosomes in non-preferential positions where alignment and biorientation are slower. Therefore, the high mis-segregation rate observed due to the high number of faulty attachments in cells with unseparated centrosomes at NEBD [bib_ref] Centrosome Dynamics as a Source of Chromosomal Instability, Nam [/bib_ref] may be related to the increase in the proportion of late-aligning polar chromosomes [fig_ref] Figure 3: Main features and models of CENP-E-mediated chromosome congression to the spindle midplane [/fig_ref]. Furthermore, a positive role of spindle elongation Additionally, pre-NEBD centrosome separation places most chromosomes between the two separated spindle poles, where alignment to the metaphase plate is rapid [bib_ref] Role of Spatial Patterns and Kinetochore Architecture in Spindle Morphogenesis, Renda [/bib_ref]. Thus, incompletely separated centrosomes increase the proportion of peripheral polar chromosomes at NEBD, consequently placing more chromosomes in non-preferential positions where alignment and biorientation are slower. Therefore, the high mis-segregation rate observed due to the high number of faulty attachments in cells with unseparated centrosomes at NEBD [bib_ref] Centrosome Dynamics as a Source of Chromosomal Instability, Nam [/bib_ref] may be related to the increase in the proportion of late-aligning polar chromosomes [fig_ref] Figure 3: Main features and models of CENP-E-mediated chromosome congression to the spindle midplane [/fig_ref]. Furthermore, a positive role of spindle elongation during alignment, biorientation, and error correction [bib_ref] The Spatial Arrangement of Chromosomes during Prometaphase Facilitates Spindle Assembly, Magidson [/bib_ref] [bib_ref] Non-Centrosomal Microtubules at Kinetochores Promote Rapid Chromosome Biorientation during Mitosis in Human..., Renda [/bib_ref] [bib_ref] The Centromere Geometry Essential for Keeping Mitosis Error Free Is Controlled by..., Lončarek [/bib_ref] implies that the congression that occurs after spindle elongation could be less efficient or more error-prone than the congression that occurs during prometaphase spindle elongation [fig_ref] Figure 3: Main features and models of CENP-E-mediated chromosome congression to the spindle midplane [/fig_ref]. This is supported by the observation that late-aligning polar chromosomes are prone to chromosome mis-segregations in HeLa cells [bib_ref] Delayed Chromosome Alignment to the Spindle Equator Increases the Rate of Chromosome..., Kuniyasu [/bib_ref]. On the other hand, another study reported that reduced prometaphase spindle elongation decreases the number of lagging chromosomes in human cells [bib_ref] A Role for Metaphase Spindle Elongation Forces in Correction of Merotelic Kinetochore..., Choi [/bib_ref] , probably due to overcharged Aurora B activity, although this phenomenon could also be related to decreased cohesion fatigue. Interestingly, it was demonstrated that unaligned polar chromosomes induce spindle positioning defects in addition to chromosome mis-segregation [bib_ref] Chromosome Misalignments Induce Spindle-Positioning Defects, Tame [/bib_ref] , suggesting a reciprocal link between aberrant spindle positioning and faulty attachments.
As polar chromosomes are characterized by their passage across the polar region, which could adversely impact their alignment compared to other chromosomes, this implies that centrosomes could be important regulators of chromosome alignment. How does the alignment of polar chromosomes occur in human cells without centrioles, the main constituents of centrosomes? Multiple analyzes showed that in cells without centrioles, spindles can bipolarize, align their chromosomes, and continuously divide [bib_ref] Centrosome-Independent Mitotic Spindle Formation in Vertebrates, Khodjakov [/bib_ref] , although at the cost of prolonged spindle assembly, chromosome mis-segregation, DNA damage, cell cycle arrest, and apoptosis [bib_ref] Human Centrosome Organization and Function in Interphase and Mitosis, Vasquez-Limeta [/bib_ref] [bib_ref] Reversible Centriole Depletion with an Inhibitor of Polo-like Kinase 4, Wong [/bib_ref] [bib_ref] Loss of Centrioles Causes Chromosomal Instability in Vertebrate Somatic Cells, Sir [/bib_ref]. The increase in the number of mis-segregations could be due to the fact that acentriolar spindles are initially disorganized and later multipolar prior to bipolarization [bib_ref] Loss of Centrioles Causes Chromosomal Instability in Vertebrate Somatic Cells, Sir [/bib_ref] , as the multipolar-to-bipolar transition is known to promote merotelic attachments and lagging chromosomes [bib_ref] A Mechanism Linking Extra Centrosomes to Chromosomal Instability, Ganem [/bib_ref]. Interestingly, after centrosome ablation in prophase, cells form functional mitotic spindles but display an increased number of syntelic polar chromosomes at the onset of anaphase [bib_ref] The Centromere Geometry Essential for Keeping Mitosis Error Free Is Controlled by..., Lončarek [/bib_ref]. Moreover, centrosome age regulates the propensity of polar chromosomes to get unaligned and mis-segregated through differential regulation of end-on attachment stability in the polar region. In conclusion, centrosomes regulate MT end-on attachments to kinetochores and chromosome congression. Consequently, their correct prepositioning is important for decreasing the proportion of polar chromosomes at NEBD, the number of erroneous attachments, and mis-segregations.
## Regulation of lateral to end-on conversion and error correction
As it was shown that kinetochores during early prometaphase are mainly captured laterally along the walls of MTs [bib_ref] The Spatial Arrangement of Chromosomes during Prometaphase Facilitates Spindle Assembly, Magidson [/bib_ref] [bib_ref] Lateral Attachment of Kinetochores to Microtubules Is Enriched in Prometaphase Rosette and..., Itoh [/bib_ref] [bib_ref] Lateral to End-on Conversion of Chromosome-Microtubule Attachment Requires Kinesins Cenp-e and MCAK, Shrestha [/bib_ref] , which sequentially turns into stable end-on attachment [bib_ref] The Spatial Arrangement of Chromosomes during Prometaphase Facilitates Spindle Assembly, Magidson [/bib_ref] [bib_ref] Spindle Assembly Checkpoint Satisfaction Occurs via End-on but Not Lateral Attachments under..., Kuhn [/bib_ref] , regulatory mechanisms of lateral to end-on transition have been a hot topic for mitosis researchers. Three main questions emerged: what signaling mechanisms bias for lateral attachment in regions where end-on attachment would be disadvantageous, such as around the spindle pole, what mechanisms ensure the transition to end-on attachment at the appropriate place and time, and what mechanisms ensue when the aforementioned mechanisms fail? First, lateral associations are presumably mediated by the motors that localize to the fibrous corona, cytoplasmic dynein, and CENP-E [bib_ref] The Kinetochore-Microtubule Interface at a Glance, Monda [/bib_ref]. Recently, the Ndc80 complex has also been implicated as a component required for lateral attachments during early mitosis in human cells [bib_ref] Lateral Attachment of Kinetochores to Microtubules Is Enriched in Prometaphase Rosette and..., Itoh [/bib_ref]. Lateral and end-on attachments are discriminated by molecular markers since only mature end-on attachments recruit components of the Astrin-SKAP complex [bib_ref] Lateral to End-on Conversion of Chromosome-Microtubule Attachment Requires Kinesins Cenp-e and MCAK, Shrestha [/bib_ref] and release SAC proteins such as Mad1 [bib_ref] Adaptive Changes in the Kinetochore Architecture Facilitate Proper Spindle Assembly, Magidson [/bib_ref] [bib_ref] Spindle Assembly Checkpoint Satisfaction Occurs via End-on but Not Lateral Attachments under..., Kuhn [/bib_ref] [bib_ref] Stable Kinetochore-Microtubule Attachment Is Sufficient to Silence the Spindle Assembly Checkpoint in..., Tauchman [/bib_ref] [fig_ref] Figure 6: Mechanisms that control the lateral to end-on transition and error correction in... [/fig_ref]. [bib_ref] Aurora Kinases and Protein Phosphatase 1 Mediate Chromosome Congression through Regulation of..., Kim [/bib_ref] [bib_ref] The Ska complex promotes Aurora B activity to ensure chromosome biorientation, Rendli [/bib_ref] [fig_ref] Figure 6: Mechanisms that control the lateral to end-on transition and error correction in... [/fig_ref] , part 2), although their contribution is not essential for the maintenance of end-on attachment in the absence of Aurora B [bib_ref] Counteraction between Astrin-PP1 and Cyclin-B-CDK1 Pathways Protects Chromosome-Microtubule Attachments Independent of Biorientation, Song [/bib_ref]. Concomitantly, the outerkinetochore checkpoint proteins BubR1, Bub1, and Mps1 that influence attachments and SAC are all removed or reduced after the formation of stable end-on attachment by the dynein-mediated stripping of the fibrous corona [bib_ref] Lateral to End-on Conversion of Chromosome-Microtubule Attachment Requires Kinesins Cenp-e and MCAK, Shrestha [/bib_ref] [bib_ref] Spindle Assembly Checkpoint Satisfaction Occurs via End-on but Not Lateral Attachments under..., Kuhn [/bib_ref] [bib_ref] The Role of Mitotic Kinases and the RZZ Complex in Kinetochore-Microtubule Attachments:..., Barbosa [/bib_ref]. 1) and (2) Simplified view of the main molecular machinery that regulates the transition from lateral (1) to end-on (2) attachment of kinetochores to microtubules. The spindle assembly checkpoint is turned on in (1) and turned off in [bib_ref] Mitotic Spindle Assembly in Animal Cells: A Fine Balancing Act, Prosser [/bib_ref]. A pool of Aurora B is present at the outer kinetochore, although the kinetochore receptor for this pool is unknown ("?"). (3) Simplified view of the main molecular machinery that regulates the correction of monotelic and syntelic attachments close to the spindle pole in the gradient of Aurora A activity (same color as the circled AurA), and (4) the correction of stretched merotelic attachments in the gradient of centromeric Aurora B activity (same color as the circled AurB) or by the Aurora B diffusing along the microtubule that originates from the distant pole (small Aurora B circles). Arrows from Aurora kinases denote phosphorylation of various kinetochore targets. Dashed arrows from kinetochore targets denote either detachment (curved arrows) or depolymerization (straight arrows) (legend in part 4). Small blue dots represent phosphorylated residues. AurA, Aurora kinase A; AurB, Aurora kinase B.
Erroneous syntelic and merotelic attachments can form during prometaphase [bib_ref] Merotelic Kinetochore Orientation Occurs Frequently during Early Mitosis in Mammalian Tissue Cells..., Cimini [/bib_ref] [bib_ref] Kinase Promotes Turnover of Kinetochore Microtubules to Reduce Chromosome Segregation Errors, Cimini [/bib_ref] and need to be corrected to avoid chromosome mis-segregation. However, since lateral attachments dominate during early spindle assembly, it is still unclear where and when erroneous attachments form during early mitotic events in human cells and what the intermediate states are [bib_ref] Merotelic Kinetochore Attachment: Causes and Effects, Gregan [/bib_ref] [bib_ref] Merotelic Kinetochore Orientation Occurs Frequently during Early Mitosis in Mammalian Tissue Cells..., Cimini [/bib_ref]. For example, monotelic attachment likely represents a normal transition to amphitelic attachment in the region between the poles [bib_ref] Chromosomes Can Congress to the Metaphase Plate before Biorientation, Kapoor [/bib_ref] , while behind the poles, this attachment would be an error that needs to be corrected [bib_ref] The Centromere Geometry Essential for Keeping Mitosis Error Free Is Controlled by..., Lončarek [/bib_ref]. Similar to lateral to end-on conversion mechanisms, a key player during error correction is also Aurora B kinase [bib_ref] Mechanisms to Avoid and Correct Erroneous Kinetochore-Microtubule Attachments, Lampson [/bib_ref] [bib_ref] The Role of Mitotic Kinases and the RZZ Complex in Kinetochore-Microtubule Attachments:..., Barbosa [/bib_ref] [bib_ref] Aurora B Tension Sensing Mechanisms in the Kinetochore Ensure Accurate Chromosome Segregation, Mcvey [/bib_ref] [bib_ref] Making the Auroras Glow: Regulation of Aurora A and B Kinase Function..., Carmena [/bib_ref]. The main activity of Aurora B in error correction involves the destabilization of incorrect attachment through the phosphorylation of several outer kinetochore proteins that directly bind to MTs, including components of the KMN network [bib_ref] Aurora B Is Enriched at Merotelic Attachment Sites, Where It Regulates MCAK, Knowlton [/bib_ref] [bib_ref] Sensing Chromosome Bi-Orientation by Spatial Separation of Aurora B Kinase from Kinetochore..., Liu [/bib_ref] [bib_ref] Kinase Promotes Turnover of Kinetochore Microtubules to Reduce Chromosome Segregation Errors, Cimini [/bib_ref] [bib_ref] Tension Promotes Kinetochore-Microtubule Release by Aurora B Kinase, Chen [/bib_ref] [bib_ref] Feedback Control in Sensing Chromosome Biorientation by the Aurora B Kinase, Salimian [/bib_ref] [bib_ref] Aurora B Phosphorylates Spatially Distinct Targets to Differentially Regulate the Kinetochore-Microtubule Interface, Welburn [/bib_ref] [bib_ref] The Ins and Outs of Aurora B Inner Centromere Localization. Front, Hindriksen [/bib_ref] [fig_ref] Figure 3: Main features and models of CENP-E-mediated chromosome congression to the spindle midplane [/fig_ref].
How does Aurora B discriminate between correct and incorrect attachments? The most appealing model is based on the low tension of erroneous attachments when compared to amphitelic ones [bib_ref] Sensing Chromosome Bi-Orientation by Spatial Separation of Aurora B Kinase from Kinetochore..., Liu [/bib_ref] [bib_ref] The Ins and Outs of Aurora B Inner Centromere Localization. Front, Hindriksen [/bib_ref] [bib_ref] A Gradient in Metaphase Tension Leads to a Scaled Cellular Response in..., Mukherjee [/bib_ref]. Such spatial positioning models of the Aurora B function are based on a physical distance between the kinase and its kinetochore substrates, either by a diffusible phosphorylation gradient or by Aurora B being positioned 1) and (2) Simplified view of the main molecular machinery that regulates the transition from lateral (1) to end-on (2) attachment of kinetochores to microtubules. The spindle assembly checkpoint is turned on in (1) and turned off in [bib_ref] Mitotic Spindle Assembly in Animal Cells: A Fine Balancing Act, Prosser [/bib_ref]. A pool of Aurora B is present at the outer kinetochore, although the kinetochore receptor for this pool is unknown ("?"). (3) Simplified view of the main molecular machinery that regulates the correction of monotelic and syntelic attachments close to the spindle pole in the gradient of Aurora A activity (same color as the circled AurA), and (4) the correction of stretched merotelic attachments in the gradient of centromeric Aurora B activity (same color as the circled AurB) or by the Aurora B diffusing along the microtubule that originates from the distant pole (small Aurora B circles). Arrows from Aurora kinases denote phosphorylation of various kinetochore targets. Dashed arrows from kinetochore targets denote either detachment (curved arrows) or depolymerization (straight arrows) (legend in part 4). Small blue dots represent phosphorylated residues. AurA, Aurora kinase A; AurB, Aurora kinase B.
The Ndc80 complex is essential for the establishment of end-on attachments of kinetochores to MTs [bib_ref] Kinetochore Microtubule Dynamics and Attachment Stability Are Regulated by Hec1, Deluca [/bib_ref] [bib_ref] The Conserved KMN Network Constitutes the Core Microtubule-Binding Site of the Kinetochore, Cheeseman [/bib_ref]. The Knl1/Mis12 complex/Ncd80 complex (KMN network) is connected to the inner kinetochore primarily by the DNA-interacting CENP-T [bib_ref] CCAN Makes Multiple Contacts with Centromeric DNA to Provide Distinct Pathways to..., Hori [/bib_ref] [bib_ref] Multiple Phosphorylations Control Recruitment of the KMN Network onto Kinetochores, Hara [/bib_ref] [bib_ref] CENP-T Provides a Structural Platform for Outer Kinetochore Assembly, Nishino [/bib_ref] [fig_ref] Figure 6: Mechanisms that control the lateral to end-on transition and error correction in... [/fig_ref]. In both yeast and human cells, Aurora B is a crucial factor involved in the control of the lateral to end-on conversion [bib_ref] Kinetochore-Microtubule Error Correction Is Driven by Differentially Regulated Interaction Modes, Kalantzaki [/bib_ref] [bib_ref] Aurora-B Kinase Pathway Controls the Lateral to End-on Conversion of Kinetochore-Microtubule Attachments..., Shrestha [/bib_ref] [fig_ref] Figure 6: Mechanisms that control the lateral to end-on transition and error correction in... [/fig_ref] , part 1). Aurora B is a component of the CPC, which includes INCENP, Borealin, and Survivin in addition to Aurora B [bib_ref] Aurora-B Kinase Pathway Controls the Lateral to End-on Conversion of Kinetochore-Microtubule Attachments..., Shrestha [/bib_ref]. Interestingly, lateral attachments are not susceptible to MT detachment mediated by the centromeric Aurora B and are not reliant on high interkinetochore tension [bib_ref] Aurora-B Kinase Pathway Controls the Lateral to End-on Conversion of Kinetochore-Microtubule Attachments..., Shrestha [/bib_ref]. Aurora B activity is counteracted by phosphatases recruited by CENP-E, Astrin complex, SKA complex, and KNL1 complex [bib_ref] CENP-E Is a Putative Kinetochore Motor That Accumulates Just before Mitosis, Yen [/bib_ref] [bib_ref] Lateral to End-on Conversion of Chromosome-Microtubule Attachment Requires Kinesins Cenp-e and MCAK, Shrestha [/bib_ref] [bib_ref] Aurora-B Kinase Pathway Controls the Lateral to End-on Conversion of Kinetochore-Microtubule Attachments..., Shrestha [/bib_ref] [bib_ref] Protein Phosphatases in the Regulation of Mitosis, Nilsson [/bib_ref] [bib_ref] Aurora Kinases and Protein Phosphatase 1 Mediate Chromosome Congression through Regulation of..., Kim [/bib_ref] [bib_ref] Human Blinkin/AF15q14 Is Required for Chromosome Alignment and the Mitotic Checkpoint through..., Kiyomitsu [/bib_ref] [bib_ref] Timely Anaphase Onset Requires a Novel Spindle and Kinetochore Complex Comprising Ska1..., Hanisch [/bib_ref]. The transition to end-on attachment is followed by the loss of outer-kinetochore associated Aurora B, leaning the balance toward the activity of BubR1-associated PP2A-B56 phosphatase, a crucial step for the establishment of end-on attachments [bib_ref] Aurora-B Kinase Pathway Controls the Lateral to End-on Conversion of Kinetochore-Microtubule Attachments..., Shrestha [/bib_ref] [fig_ref] Figure 2: Mechanisms of kinetochore search-and-capture and gathering on the spindle [/fig_ref]. Further enrichment of the Astrin complex on end-on tethered kinetochores, along with physical separation of centromere-associated Aurora B from outer-kinetochore substrates [bib_ref] Sensing Chromosome Bi-Orientation by Spatial Separation of Aurora B Kinase from Kinetochore..., Liu [/bib_ref] , facilitates the maintenance of mature end-on attachments [bib_ref] Aurora-B Kinase Pathway Controls the Lateral to End-on Conversion of Kinetochore-Microtubule Attachments..., Shrestha [/bib_ref] [fig_ref] Figure 2: Mechanisms of kinetochore search-and-capture and gathering on the spindle [/fig_ref]. Furthermore, Astrin stabilizes monotelic attachments by opposing attachment destabilization, primarily through its role in delivering PP1 phosphatase to the outer kinetochore [fig_ref] Figure 6: Mechanisms that control the lateral to end-on transition and error correction in... [/fig_ref] , part 2), thus opposing CDK1 kinase activity [bib_ref] Counteraction between Astrin-PP1 and Cyclin-B-CDK1 Pathways Protects Chromosome-Microtubule Attachments Independent of Biorientation, Song [/bib_ref] , together with coinciding changes in the kinetochore architecture [bib_ref] Dynamic Kinetochore Size Regulation Promotes Microtubule Capture and Chromosome Biorientation in Mitosis, Sacristan [/bib_ref]. Thus, in the absence of Astrin, end-on attachments form but are not stably maintained [bib_ref] Aurora-B Kinase Pathway Controls the Lateral to End-on Conversion of Kinetochore-Microtubule Attachments..., Shrestha [/bib_ref] [bib_ref] Counteraction between Astrin-PP1 and Cyclin-B-CDK1 Pathways Protects Chromosome-Microtubule Attachments Independent of Biorientation, Song [/bib_ref]. CENP-E and Ska complex also deliver pools of PP1 to the outer kinetochore during the end-on conversion, which counteracts attachment destabilization [bib_ref] Aurora Kinases and Protein Phosphatase 1 Mediate Chromosome Congression through Regulation of..., Kim [/bib_ref] [bib_ref] The Ska complex promotes Aurora B activity to ensure chromosome biorientation, Rendli [/bib_ref] [fig_ref] Figure 6: Mechanisms that control the lateral to end-on transition and error correction in... [/fig_ref] , part 2), although their contribution is not essential for the maintenance of end-on attach-Cells 2022, 11, 1531 13 of 27 ment in the absence of Aurora B [bib_ref] Counteraction between Astrin-PP1 and Cyclin-B-CDK1 Pathways Protects Chromosome-Microtubule Attachments Independent of Biorientation, Song [/bib_ref]. Concomitantly, the outer-kinetochore checkpoint proteins BubR1, Bub1, and Mps1 that influence attachments and SAC are all removed or reduced after the formation of stable end-on attachment by the dynein-mediated stripping of the fibrous corona [bib_ref] Lateral to End-on Conversion of Chromosome-Microtubule Attachment Requires Kinesins Cenp-e and MCAK, Shrestha [/bib_ref] [bib_ref] Spindle Assembly Checkpoint Satisfaction Occurs via End-on but Not Lateral Attachments under..., Kuhn [/bib_ref] [bib_ref] The Role of Mitotic Kinases and the RZZ Complex in Kinetochore-Microtubule Attachments:..., Barbosa [/bib_ref].
Erroneous syntelic and merotelic attachments can form during prometaphase [bib_ref] Merotelic Kinetochore Orientation Occurs Frequently during Early Mitosis in Mammalian Tissue Cells..., Cimini [/bib_ref] [bib_ref] Kinase Promotes Turnover of Kinetochore Microtubules to Reduce Chromosome Segregation Errors, Cimini [/bib_ref] and need to be corrected to avoid chromosome mis-segregation. However, since lateral attachments dominate during early spindle assembly, it is still unclear where and when erroneous attachments form during early mitotic events in human cells and what the intermediate states are [bib_ref] Merotelic Kinetochore Attachment: Causes and Effects, Gregan [/bib_ref] [bib_ref] Merotelic Kinetochore Orientation Occurs Frequently during Early Mitosis in Mammalian Tissue Cells..., Cimini [/bib_ref]. For example, monotelic attachment likely represents a normal transition to amphitelic attachment in the region between the poles [bib_ref] Chromosomes Can Congress to the Metaphase Plate before Biorientation, Kapoor [/bib_ref] , while behind the poles, this attachment would be an error that needs to be corrected [bib_ref] The Centromere Geometry Essential for Keeping Mitosis Error Free Is Controlled by..., Lončarek [/bib_ref]. Similar to lateral to end-on conversion mechanisms, a key player during error correction is also Aurora B kinase [bib_ref] Mechanisms to Avoid and Correct Erroneous Kinetochore-Microtubule Attachments, Lampson [/bib_ref] [bib_ref] The Role of Mitotic Kinases and the RZZ Complex in Kinetochore-Microtubule Attachments:..., Barbosa [/bib_ref] [bib_ref] Aurora B Tension Sensing Mechanisms in the Kinetochore Ensure Accurate Chromosome Segregation, Mcvey [/bib_ref] [bib_ref] Making the Auroras Glow: Regulation of Aurora A and B Kinase Function..., Carmena [/bib_ref]. The main activity of Aurora B in error correction involves the destabilization of incorrect attachment through the phosphorylation of several outer kinetochore proteins that directly bind to MTs, including components of the KMN network [bib_ref] Aurora B Is Enriched at Merotelic Attachment Sites, Where It Regulates MCAK, Knowlton [/bib_ref] [bib_ref] Sensing Chromosome Bi-Orientation by Spatial Separation of Aurora B Kinase from Kinetochore..., Liu [/bib_ref] [bib_ref] Kinase Promotes Turnover of Kinetochore Microtubules to Reduce Chromosome Segregation Errors, Cimini [/bib_ref] [bib_ref] Tension Promotes Kinetochore-Microtubule Release by Aurora B Kinase, Chen [/bib_ref] [bib_ref] Feedback Control in Sensing Chromosome Biorientation by the Aurora B Kinase, Salimian [/bib_ref] [bib_ref] Aurora B Phosphorylates Spatially Distinct Targets to Differentially Regulate the Kinetochore-Microtubule Interface, Welburn [/bib_ref] [bib_ref] The Ins and Outs of Aurora B Inner Centromere Localization. Front, Hindriksen [/bib_ref] [fig_ref] Figure 6: Mechanisms that control the lateral to end-on transition and error correction in... [/fig_ref] , parts 3 and 4).
How does Aurora B discriminate between correct and incorrect attachments? The most appealing model is based on the low tension of erroneous attachments when compared to amphitelic ones [bib_ref] Sensing Chromosome Bi-Orientation by Spatial Separation of Aurora B Kinase from Kinetochore..., Liu [/bib_ref] [bib_ref] The Ins and Outs of Aurora B Inner Centromere Localization. Front, Hindriksen [/bib_ref] [bib_ref] A Gradient in Metaphase Tension Leads to a Scaled Cellular Response in..., Mukherjee [/bib_ref]. Such spatial positioning models of the Aurora B function are based on a physical distance between the kinase and its kinetochore substrates, either by a diffusible phosphorylation gradient or by Aurora B being positioned on a long tether [bib_ref] The Role of Mitotic Kinases and the RZZ Complex in Kinetochore-Microtubule Attachments:..., Barbosa [/bib_ref]. However, low interkinetochore distances do not induce Aurora B-mediated error correction [bib_ref] Complete Microtubule-Kinetochore Occupancy Favours the Segregation of Merotelic Attachments, Dudka [/bib_ref] , decreased phosphorylation of incorrect attachments can ensue, regardless of their tension state [bib_ref] The Binding of Borealin to Microtubules Underlies a Tension Independent Kinetochore-Microtubule Error..., Trivedi [/bib_ref] , and amphitelic attachments can form in cells with a kinetochore-proximal pool of Aurora B if cohesion is stabilized [bib_ref] Untangling the Contribution of Haspin and Bub1 to Aurora B Function during..., Hadders [/bib_ref] [bib_ref] Inner Centromere Localization of the CPC Maintains Centromere Cohesion and Allows Mitotic..., Hengeveld [/bib_ref]. Therefore, it was suggested that the inner centromere localization of Aurora B is not a prerequisite for the phosphorylation of erroneous kinetochore-MT attachments nor for the stabilization of correct attachments [bib_ref] The Ins and Outs of Aurora B Inner Centromere Localization. Front, Hindriksen [/bib_ref] , although it is crucial for the regulation of MCAK activity to destabilize MTs [bib_ref] Aurora B Is Enriched at Merotelic Attachment Sites, Where It Regulates MCAK, Knowlton [/bib_ref] [bib_ref] The Right Place at the Right Time: Aurora B Kinase Localization to..., Broad [/bib_ref]. Recently, distinct populations of Aurora B were found localized to the inner centromere, outer centromere, and outer kinetochore, although the receptor for Aurora B at the outer kinetochore is unknown [bib_ref] Dynamic Kinetochore Size Regulation Promotes Microtubule Capture and Chromosome Biorientation in Mitosis, Sacristan [/bib_ref]. While centromeric Aurora B appears necessary for error correction, intriguingly, it is not required for phosphorylation of kinetochore substrates, which presumably depends only on Aurora B localized to the kinetochore [bib_ref] Untangling the Contribution of Haspin and Bub1 to Aurora B Function during..., Hadders [/bib_ref] [bib_ref] Centromere-Localized Aurora B Kinase Is Required for the Fidelity of Chromosome Segregation, Liang [/bib_ref] [bib_ref] Aurora B Kinase Is Recruited to Multiple Discrete Kinetochore and Centromere Regions..., Broad [/bib_ref]. Once correct kinetochore-MT attachments are formed, and tension is established, the Aurora B pool at kinetochores is presumably lost [bib_ref] Dynamic Kinetochore Size Regulation Promotes Microtubule Capture and Chromosome Biorientation in Mitosis, Sacristan [/bib_ref]. Further mechanisms that could discriminate different attachments include progressive restriction of attachment geometry [bib_ref] Dynamic Kinetochore Size Regulation Promotes Microtubule Capture and Chromosome Biorientation in Mitosis, Sacristan [/bib_ref] [bib_ref] Congressing Kinetochores Progressively Load Ska Complexes to Prevent Force-Dependent Detachment, Auckland [/bib_ref] and MT-pulling or tension-associated active detachment [bib_ref] Tension Directly Stabilizes Reconstituted Kinetochore-Microtubule Attachments, Akiyoshi [/bib_ref].
How exactly does Aurora B mediate the destabilization of incorrect attachments? Aurora B phosphorylation could promote both detachment and depolymerization of endon attached MTs [bib_ref] Correcting Improper Chromosome-Spindle Attachments during Cell Division, Lampson [/bib_ref] [bib_ref] The Conserved KMN Network Constitutes the Core Microtubule-Binding Site of the Kinetochore, Cheeseman [/bib_ref] [bib_ref] Kinase Promotes Turnover of Kinetochore Microtubules to Reduce Chromosome Segregation Errors, Cimini [/bib_ref] [bib_ref] Aurora B Phosphorylates Spatially Distinct Targets to Differentially Regulate the Kinetochore-Microtubule Interface, Welburn [/bib_ref] [fig_ref] Figure 6: Mechanisms that control the lateral to end-on transition and error correction in... [/fig_ref]. Recent experiments indicate that Aurora B promotes detachment under high tension and depolymerization under low tension [bib_ref] Tension Promotes Kinetochore-Microtubule Release by Aurora B Kinase, Chen [/bib_ref]. Therefore, tension is probably not an input for error correction, but it regulates downstream response to Aurora B phosphorylation. This could be important in distinguishing lowtension syntelic attachments from merotelic attachments that are usually stretched [bib_ref] Aurora B Is Enriched at Merotelic Attachment Sites, Where It Regulates MCAK, Knowlton [/bib_ref] [bib_ref] Sensing Chromosome Bi-Orientation by Spatial Separation of Aurora B Kinase from Kinetochore..., Liu [/bib_ref] [fig_ref] Figure 6: Mechanisms that control the lateral to end-on transition and error correction in... [/fig_ref]. The major unresolved question is how Aurora B leaves the correct attachments unaffected but corrects the stretched merotelic attachments. This could be because at a merotelic kinetochore, only the attachments of MTs emanating from the distal pole are presumably within the centromeric Aurora B phosphorylation gradient and will be destabilized, whereas the correct attachments will be unaffected [fig_ref] Figure 4: Mechanisms that set the distance between the chromosome and the spindle pole... [/fig_ref]. Moreover, a recent model proposed that MTs from the distal pole provide a path for Aurora B diffusion because they pass close to the centromere; thus, the active kinase grasps only erroneous attachments without affecting the ends of MTs from the proximal pole [bib_ref] The Binding of Borealin to Microtubules Underlies a Tension Independent Kinetochore-Microtubule Error..., Trivedi [/bib_ref] [fig_ref] Figure 6: Mechanisms that control the lateral to end-on transition and error correction in... [/fig_ref] , part 4). Regarding Aurora B-mediated MT depolymerization that brings kinetochores with syntelic attachment to spindle pole [bib_ref] Correcting Improper Chromosome-Spindle Attachments during Cell Division, Lampson [/bib_ref] , it was proposed that this could be followed by MT detachment mediated by pole-localized Aurora A that also phosphorylates the Ndc80 complex [bib_ref] A Kinase Contributes to a Pole-Based Error Correction Pathway, Ye [/bib_ref] [bib_ref] Aurora A Kinase Phosphorylates Hec1 to Regulate Metaphase Kinetochore-Microtubule Dynamics, Deluca [/bib_ref] [fig_ref] Figure 3: Main features and models of CENP-E-mediated chromosome congression to the spindle midplane [/fig_ref]. Although Aurora A is mostly localized around spindle poles [bib_ref] Making the Auroras Glow: Regulation of Aurora A and B Kinase Function..., Carmena [/bib_ref] , it has recently been connected even with error correction of aligned chromosomes promoted by chromosome oscillations [bib_ref] Chromosome Oscillation Promotes Aurora A-Dependent Hec1 Phosphorylation and Mitotic Fidelity, Iemura [/bib_ref].
In addition to their role in the regulation of the stability and dynamics of MTs, Aurora kinases are also involved in the regulation of the main kinetochore motors involved in the congression. For example, phosphorylation of CENP-E near its motor domain by Aurora A and Aurora B is necessary for the congression of polar chromosomes [bib_ref] Aurora Kinases and Protein Phosphatase 1 Mediate Chromosome Congression through Regulation of..., Kim [/bib_ref] [fig_ref] Figure 6: Mechanisms that control the lateral to end-on transition and error correction in... [/fig_ref]. Interestingly, phosphorylation of CENP-E by Aurora kinases reduces its affinity for MT, and it is not yet clear how reducing MT affinity would increase the processivity of CENP-E required for polar chromosome congression. Dynein, on the other hand, is regulated by Plk1 phosphorylation in a chromosome position-dependent manner [bib_ref] Phosphorylation Regulates Targeting of Cytoplasmic Dynein to Kinetochores during Mitosis, Whyte [/bib_ref] [bib_ref] Polo-like Kinase1 Is Required for Recruitment of Dynein to Kinetochores during Mitosis, Bader [/bib_ref] but is also indirectly controlled by Aurora B [bib_ref] Mitotic Control of Kinetochore-Associated Dynein and Spindle Orientation by Human Spindly, Ying [/bib_ref] [bib_ref] Zwint-1 Is a Novel Aurora B Substrate Required for the Assembly of..., Kasuboski [/bib_ref]. Interestingly, CENP-E is known to counteract Aurora-B mediated Ncd80 phosphorylation, as inhibition of CENP-E increases the phosphorylation of Ncd80 on polar chromosomes in a tensionindependent manner [bib_ref] A Tension-Independent Mechanism Reduces Aurora B-Mediated Phosphorylation upon Microtubule Capture by CENP-E..., Taveras [/bib_ref]. Furthermore, Aurora B inhibition induces the removal of Mad2 from polar chromosomes after CENP-E depletion [bib_ref] Kinetochore Motors Drive Congression of Peripheral Polar Chromosomes by Overcoming Random Arm-Ejection..., Barisic [/bib_ref] [bib_ref] Kinase Cooperates with CENP-E to Promote Timely Anaphase Onset, Maia [/bib_ref] , suggesting that CENP-E could oppose the effects of Aurora B on MT destabilization, possibly by its role in PP1 delivery. Clearly, more work is needed to unravel the complex coordination between Aurora kinases and the main motor proteins involved in the congression of polar chromosomes, especially in different spindle regions and at different time points during early mitosis.
## Mis-segregation and aneuploidy of polar chromosomes
## Different ways to mis-segregation through polar chromosomes
There are multiple possible pathways by which unaligned polar chromosomes can lead to mis-segregation and aneuploidy [fig_ref] Figure 7: Routes to chromosome mis-segregation through unaligned polar chromosomes and their consequences [/fig_ref]. First, a polar chromosome can remain unaligned through metaphase and anaphase and mis-segregate while still stuck at the polar region [fig_ref] Figure 7: Routes to chromosome mis-segregation through unaligned polar chromosomes and their consequences [/fig_ref]. Interestingly, the frequencies of unaligned chromosomes in anaphase across different cell types are not known, although they are expected to be low [bib_ref] A Mechanism Linking Extra Centrosomes to Chromosomal Instability, Ganem [/bib_ref] [bib_ref] Chromosome Length and Gene Density Contribute to Micronuclear Membrane Stability, Mammel [/bib_ref]. This is because cells have intricate systems to avoid the occurrence of unaligned chromosomes, but if such chromosomes persist, they must pass the control of SAC [fig_ref] Figure 7: Routes to chromosome mis-segregation through unaligned polar chromosomes and their consequences [/fig_ref] , route 1). Although SAC activity can be weakened, which would allow mis-segregation of unaligned polar chromosomes by precocious anaphase start [bib_ref] Chromosomes Trapped in Micronuclei Are Liable to Segregation Errors, Soto [/bib_ref] , from extensive data, it is becoming clear that SAC-related deficiencies are rare in human tumors, and even cells with high CIN generally do not enter anaphase precociously [bib_ref] The Impact of Mitotic Errors on Cell Proliferation and Tumorigenesis, Levine [/bib_ref] [bib_ref] Causes and Consequences of Aneuploidy in Cancer, Gordon [/bib_ref]. However, attenuation of SAC promotes aneuploidy [bib_ref] Prediction Model for Aneuploidy in Early Human Embryo Development Revealed by Single-Cell..., Vera-Rodriguez [/bib_ref] , weakened SAC is associated with certain aneuploidies during early embryogenesis [bib_ref] Cell-Size-Independent Spindle Checkpoint Failure Underlies Chromosome Segregation Error in Mouse Embryos, Vázquez-Diez [/bib_ref] , and rare genetic disorders with altered SAC, such as mosaic variegated aneuploidy (MVA), are documented [bib_ref] Biallelic TRIP13 Mutations Predispose to Wilms Tumor and Chromosome Missegregation, Yost [/bib_ref]. Although the level of SAC response is understudied in unperturbed systems, much more is known about the SAC response after different perturbations. For example, several studies have revealed that SAC is not an all-or-nothing response in human cells, but it scales with the number of unattached kinetochores [bib_ref] The Spindle Assembly Checkpoint Works like a Rheostat Rather than a Toggle..., Collin [/bib_ref] [bib_ref] Kinetic Framework of Spindle Assembly Checkpoint Signalling, Dick [/bib_ref] , delaying anaphase onset for up to a few hours. Unaligned chromosomes produced similar mitotic delays in Ptk1 cells [bib_ref] The Checkpoint Delaying Anaphase in Response to Chromosome Monoorientation Is Mediated by..., Rieder [/bib_ref]. These findings imply that only a low number of unattached polar chromosomes could induce moderate mitotic delays, which would lead to mis-segregation of unaligned chromosomes.
more is known about the SAC response after different perturbations. For example, several studies have revealed that SAC is not an all-or-nothing response in human cells, but it scales with the number of unattached kinetochores [bib_ref] The Spindle Assembly Checkpoint Works like a Rheostat Rather than a Toggle..., Collin [/bib_ref] [bib_ref] Kinetic Framework of Spindle Assembly Checkpoint Signalling, Dick [/bib_ref] , delaying anaphase onset for up to a few hours. Unaligned chromosomes produced similar mitotic delays in Ptk1 cells [bib_ref] The Checkpoint Delaying Anaphase in Response to Chromosome Monoorientation Is Mediated by..., Rieder [/bib_ref]. These findings imply that only a low number of unattached polar chromosomes could induce moderate mitotic delays, which would lead to mis-segregation of unaligned chromosomes. Accordingly, studies that used CENP-E perturbations reported mis-segregation of one or a few unaligned polar chromosomes after moderate mitotic delays in HeLa [bib_ref] Kinase Cooperates with CENP-E to Promote Timely Anaphase Onset, Maia [/bib_ref] , RPE1 [bib_ref] Impaired CENP-E Function Renders Large Chromosomes More Vulnerable to Congression Failure, Tovini [/bib_ref] [bib_ref] Endomembranes promote chromosome missegregation by ensheathing misaligned chromosomes, Ferrandiz [/bib_ref] , and mouse embryonic cells [bib_ref] Aneuploidy Acts Both Oncogenically and as a Tumor Suppressor, Weaver [/bib_ref]. A similar phenomenon was observed after treatment of cells with nanomolar doses of nocodazole [bib_ref] Kinetic Framework of Spindle Assembly Checkpoint Signalling, Dick [/bib_ref]. Interestingly, the SAC response was robust since Mad1 was recruited on unaligned kinetochores in such cells, and mitotic delays were at the level of a few hours. Moreover, anaphase onset was not the result of 'cohesion fatigue' or 'mitotic slippage' [bib_ref] Cohesion Fatigue Induces Chromatid Separation in Cells Delayed at Metaphase, Daum [/bib_ref] [bib_ref] Mitotic Checkpoint Slippage in Humans Occurs via Cyclin B Destruction in the..., Brito [/bib_ref] since it was preceded by the loss of Mad proteins from unaligned kinetochores, implying that both sister kinetochores acquired endon connections that were able to satisfy the SAC [bib_ref] Kinase Cooperates with CENP-E to Promote Timely Anaphase Onset, Maia [/bib_ref] [bib_ref] Kinetic Framework of Spindle Assembly Checkpoint Signalling, Dick [/bib_ref] [bib_ref] Impaired CENP-E Function Renders Large Chromosomes More Vulnerable to Congression Failure, Tovini [/bib_ref] [bib_ref] Endomembranes promote chromosome missegregation by ensheathing misaligned chromosomes, Ferrandiz [/bib_ref]. However, it is unclear what types of attachment could satisfy the SAC at the pole since syntelic, monotelic, and lateral attachments induce a large SAC response [bib_ref] Stable Kinetochore-Microtubule Attachment Is Sufficient to Silence the Spindle Assembly Checkpoint in..., Tauchman [/bib_ref] , although it is possible that the SAC signal wears faster over time if stable end-on attachments are present at one of the sister kinetochores [bib_ref] The Centromere Geometry Essential for Keeping Mitosis Error Free Is Controlled by..., Lončarek [/bib_ref] [bib_ref] Kinetochore-Microtubule Attachment Is Sufficient to Satisfy the Human Spindle Assembly Checkpoint, Etemad [/bib_ref]. A recent study presented an intriguing model in which difficulties associated with the inability of unaligned chromosomes to congress are not necessarily caused by defects related to MTs, SAC, or motors but rather due to the presence of a complex system of organelle remnants behind the spindle poles, termed endomembranes, which could ensheat polar chromosomes and prevent their efficient capture by MTs, resulting in aneuploidy [bib_ref] Endomembranes promote chromosome missegregation by ensheathing misaligned chromosomes, Ferrandiz [/bib_ref]. However, for definitive conclusions about the mechanisms underlying unaligned chromosome mis-segregation in unperturbed cells, one would need to track the origin and fate of mis-segregations through whole mitosis, similar to what was done recently from metaphase to telophase in human cells [bib_ref] Kinetochore Life Histories Reveal an Aurora-B-Dependent Error Correction Mechanism in Anaphase, Sen [/bib_ref].
Mis-segregation of unaligned chromosomes could induce extra chromosomes in the main nuclei or the formation of micronuclei in one of the daughter cells [bib_ref] Chromosomes Trapped in Micronuclei Are Liable to Segregation Errors, Soto [/bib_ref] [bib_ref] Nuclear Envelope Assembly Defects Link Mitotic Errors to Chromothripsis, Liu [/bib_ref] [fig_ref] Figure 7: Routes to chromosome mis-segregation through unaligned polar chromosomes and their consequences [/fig_ref] , route 1), where the latter is particularly detrimental to genome stability as it is related to chromothripsis [bib_ref] Chromothripsis from DNA Damage in Micronuclei, Zhang [/bib_ref]. Interestingly, the micronucleus generated from the polar chromosome is different from the one generated by a lagging chromosome stuck in the cleavage furrow, as nuclear lamina defects and the response to DNA damage are not pronounced in micronuclei that originate from polar chromosomes [bib_ref] Nuclear Envelope Assembly Defects Link Mitotic Errors to Chromothripsis, Liu [/bib_ref] [fig_ref] Figure 7: Routes to chromosome mis-segregation through unaligned polar chromosomes and their consequences [/fig_ref]. This phenomenon could drastically affect the propensity to propagate unaligned chromosomes over generations. However, a recent study indicated that micronuclear stability is determined more by the identity and length of chromosomes trapped within the micronucleus than by their mis-segregation position [bib_ref] Chromosome Length and Gene Density Contribute to Micronuclear Membrane Stability, Mammel [/bib_ref]. Regardless of the mechanisms, if an unaligned polar chromosome satisfies the SAC and results in aneuploidy, it is expected that daughter cells would struggle in proliferation independently of aneuploidy, as prolonged prometaphase activates p53-p21 dependent apoptotic response that blocks further daughter cell proliferation [bib_ref] Prolonged Prometaphase Blocks Daughter Cell Proliferation despite Normal Completion of Mitosis, Uetake [/bib_ref] [bib_ref] Chromosome Mis-Segregation Generates Cell-Cycle-Arrested Cells with Complex Karyotypes That Are Eliminated by..., Santaguida [/bib_ref]. The monosomic daughter cell would be at an even higher risk of cell cycle arrest since, in human somatic cells, chromosome loss impairs proliferation and genomic stability more than chromosome trisomy [bib_ref] Systems Approaches Identify the Consequences of Monosomy in Somatic Human Cells, Chunduri [/bib_ref]. Recently, it was shown that even shortened mitosis induced upon SAC inhibition increased the apoptotic response in human cells [bib_ref] Separase-triggered apoptosis enforces minimal length of mitosis, Hellmuth [/bib_ref]. Overall, we speculate that among the mis-segregations caused by polar chromosomes, selection would favor those that are able to satisfy the SAC in the well-defined time frame [fig_ref] Figure 7: Routes to chromosome mis-segregation through unaligned polar chromosomes and their consequences [/fig_ref].
Second, even if the polar chromosomes align at the metaphase plate, there is still a possibility that their attachments could be erroneous. The most studied type of erroneous attachment is the merotelic attachment [bib_ref] Merotelic Kinetochore Attachment: Causes and Effects, Gregan [/bib_ref] [fig_ref] Figure 7: Routes to chromosome mis-segregation through unaligned polar chromosomes and their consequences [/fig_ref] , route 2). As kinetochores in late prometaphase and metaphase cells are oriented parallel to the main spindle axis [bib_ref] The Spatial Arrangement of Chromosomes during Prometaphase Facilitates Spindle Assembly, Magidson [/bib_ref] [bib_ref] Adaptive Changes in the Kinetochore Architecture Facilitate Proper Spindle Assembly, Magidson [/bib_ref] , this back-to-back orientation minimizes the chance that the kinetochore captures MTs from the distant pole [bib_ref] The Centromere Geometry Essential for Keeping Mitosis Error Free Is Controlled by..., Lončarek [/bib_ref]. This is, however, not the case with polar chromosomes, as during early prometaphase, polar chromosomes are characterized by random orientations with respect to the main spindle axis [bib_ref] The Spatial Arrangement of Chromosomes during Prometaphase Facilitates Spindle Assembly, Magidson [/bib_ref]. Thus, are polar chromosomes more susceptible to merotelic attachments? There are prominent mechanisms that ensure kinetochores avoid stable attachment near the pole and mechanisms that correct such attachments if they occur (see Sections 4.3 and 5). However, because of their proximity to the pole, it is conceivable that polar chromosomes could have a higher risk of acquiring syntelic attachments, which may convert to merotelic, although such a hypothesis requires experimental testing. Interestingly, in a photoactivation study in HeLa cells, it was reported that late-aligning polar chromosomes increase the rate of lagging chromosomes [bib_ref] Delayed Chromosome Alignment to the Spindle Equator Increases the Rate of Chromosome..., Kuniyasu [/bib_ref] , and lagging chromosomes are mostly associated with merotelic attachments [bib_ref] Merotelic Kinetochore Orientation Occurs Frequently during Early Mitosis in Mammalian Tissue Cells..., Cimini [/bib_ref].
If merotelic attachments are formed and persist until anaphase onset, this does not necessarily imply aneuploidy. In a set of recent studies, it was reported that the Aurora B midzone gradient mediates phosphorylation of outer kinetochore proteins even during anaphase [bib_ref] The Aurora B Gradient Sustains Kinetochore Stability in Anaphase, Papini [/bib_ref] , at similar sites as in pre-anaphase cells [bib_ref] Aurora B Phosphorylates Spatially Distinct Targets to Differentially Regulate the Kinetochore-Microtubule Interface, Welburn [/bib_ref]. Thus, it seems that error correction mediated by Aurora B has an additional layer operating in early anaphase [bib_ref] Kinetochore Life Histories Reveal an Aurora-B-Dependent Error Correction Mechanism in Anaphase, Sen [/bib_ref] [bib_ref] An Anaphase Surveillance Mechanism Prevents Micronuclei Formation from Frequent Chromosome Segregation Errors, Orr [/bib_ref] [fig_ref] Figure 7: Routes to chromosome mis-segregation through unaligned polar chromosomes and their consequences [/fig_ref] , route 2), which could explain previous observations that the proportion of lagging chromosomes during anaphase is by an order of magnitude higher than the proportion of cells with aneuploidy in the same population [bib_ref] Chromosome Missegregation in Human Cells Arises through Specific Types of Kinetochore-Microtubule Attachment..., Thompson [/bib_ref]. Furthermore, this led to a redefinition of lagging kinetochores by introducing a new term of 'lazy' kinetochores, transiently lagging kinetochores that are quickly and efficiently corrected during the early anaphase by the Aurora B-dependent mechanism [bib_ref] Kinetochore Life Histories Reveal an Aurora-B-Dependent Error Correction Mechanism in Anaphase, Sen [/bib_ref] [fig_ref] Figure 7: Routes to chromosome mis-segregation through unaligned polar chromosomes and their consequences [/fig_ref] , route 2). However, in certain situations, even moderate alignment defects can induce malfunctioning nuclear morphologies and micronuclei [bib_ref] Mitotic Chromosome Alignment Ensures Mitotic Fidelity by Promoting Interchromosomal Compaction during Anaphase, Fonseca [/bib_ref] , calling for long-term tracking of chromosome fates during mitosis.
Third, even if polar chromosomes align at the metaphase plate, they could be characterized by various types of alignment problems compared to other chromosomes. For example, late-aligning polar chromosomes could have perturbed end-on attachment stability because global MT stability increases as the cell progresses from prometaphase to metaphase [bib_ref] Cyclin A Regulates Kinetochore Microtubules to Promote Faithful Chromosome Segregation, Kabeche [/bib_ref]. As out-of-plate movements are extremely rarely observed in human non-transformed cells [bib_ref] The Spatial Arrangement of Chromosomes during Prometaphase Facilitates Spindle Assembly, Magidson [/bib_ref] , instability of position within the metaphase plate could be particularly pronounced in tumor cells due to the defective stability of kinetochore MTs [fig_ref] Figure 3: Main features and models of CENP-E-mediated chromosome congression to the spindle midplane [/fig_ref] , a characteristic trait of most chromosomally unstable tumors [bib_ref] Deviant Kinetochore Microtubule Dynamics Underlie Chromosomal Instability, Bakhoum [/bib_ref] [bib_ref] Genome Stability Is Ensured by Temporal Control of Kinetochore-Microtubule Dynamics, Bakhoum [/bib_ref]. Tumor systems are expected to have a larger fraction of polar chromosomes at NEBD [bib_ref] Kinetochore Motors Drive Congression of Peripheral Polar Chromosomes by Overcoming Random Arm-Ejection..., Barisic [/bib_ref] [bib_ref] Delayed Chromosome Alignment to the Spindle Equator Increases the Rate of Chromosome..., Kuniyasu [/bib_ref] [bib_ref] Impaired CENP-E Function Renders Large Chromosomes More Vulnerable to Congression Failure, Tovini [/bib_ref]. Overall, we speculate that the stability of chromosomes within the plate may depend on the time of chromosome arrival at the equator. Instability of chromosome position within the plate would put a burden on the mitotic fidelity by increasing the number of unaligned chromosomes, thus triggering a loop of defects associated with these chromosomes [fig_ref] Figure 7: Routes to chromosome mis-segregation through unaligned polar chromosomes and their consequences [/fig_ref] , route 3). In this feedback loop, late-aligning chromosomes are frequently expelled from the pseudo-metaphase plate back to the poles, where they must avoid both formations of merotelic attachments and premature satisfaction of SAC [fig_ref] Figure 7: Routes to chromosome mis-segregation through unaligned polar chromosomes and their consequences [/fig_ref]. Consistent with this assumption, unaligned chromosomes are frequently observed in systems that are characterized by a higher rate of lagging chromosomes and defective stability of MT, such as immature mouse organoids [bib_ref] Chromosome Segregation Fidelity in Epithelia Requires Tissue Architecture, Knouse [/bib_ref] and human cells treated with nanomolar doses of nocodazole [bib_ref] Adaptive Changes in the Kinetochore Architecture Facilitate Proper Spindle Assembly, Magidson [/bib_ref] [bib_ref] Kinetic Framework of Spindle Assembly Checkpoint Signalling, Dick [/bib_ref]. Furthermore, unaligned chromosomes are not efficiently phosphorylated by Aurora B in tumor cells, contrary to non-transformed cells [bib_ref] Feedback Control in Sensing Chromosome Biorientation by the Aurora B Kinase, Salimian [/bib_ref] , which could explain their inefficient correction in tumor systems.
Altogether, polar chromosomes may be prone to various types of mis-segregations and alignment problems that could deleteriously affect mitotic fidelity, especially in tumor cells with perturbed MT stability and error correction mechanisms. However, it is currently unclear if chromosome mis-segregations and whole-chromosome aneuploidies are biased and to what extent toward certain chromosomes, with much evidence supporting [bib_ref] Impaired CENP-E Function Renders Large Chromosomes More Vulnerable to Congression Failure, Tovini [/bib_ref] [bib_ref] Non-Random Mis-Segregation of Human Chromosomes, Worrall [/bib_ref] [bib_ref] Chromosome Segregation Is Biased by Kinetochore Size, Drpic [/bib_ref] [bib_ref] Human Chromosome-Specific Aneuploidy Is Influenced by DNA-Dependent Centromeric Features, Dumont [/bib_ref] [bib_ref] DNA Sequence-Specific Binding of CENP-B Enhances the Fidelity of Human Centromere Function, Fachinetti [/bib_ref] and some discouraging [bib_ref] Aneuploidy in Mitosis of PtK1 Cells Is Generated by Random Loss and..., Torosantucci [/bib_ref] such a hypothesis in vertebrate model systems after different spindle perturbations. The reported biases are related to the size of the chromosome or kinetochore [bib_ref] Chromosome Length and Gene Density Contribute to Micronuclear Membrane Stability, Mammel [/bib_ref] [bib_ref] Non-Random Mis-Segregation of Human Chromosomes, Worrall [/bib_ref] [bib_ref] Chromosome Segregation Is Biased by Kinetochore Size, Drpic [/bib_ref] , the duration of prometaphase and cohesion fatigue [bib_ref] Non-Random Mis-Segregation of Human Chromosomes, Worrall [/bib_ref] , or the level of centromere-associated proteins [bib_ref] Human Chromosome-Specific Aneuploidy Is Influenced by DNA-Dependent Centromeric Features, Dumont [/bib_ref] [bib_ref] DNA Sequence-Specific Binding of CENP-B Enhances the Fidelity of Human Centromere Function, Fachinetti [/bib_ref].
## Polar chromosomes in cancer-aneuploidy and cin
Aneuploidy is a hallmark of human cancers and is tightly interlinked with high rates of chromosome mis-segregation and CIN [bib_ref] Context Is Everything: Aneuploidy in Cancer, Ben-David [/bib_ref]. Chromosome congression and alignment defects are reported in human tumors with high CIN rates [bib_ref] Mechanisms of Chromosome Congression during Mitosis, Maiato [/bib_ref] [bib_ref] Causes and Consequences of Aneuploidy in Cancer, Gordon [/bib_ref] , although the relevance of such observations for tumorigenesis is unclear, as there are many genetic disruptions that could lead to this phenotype. Current evidence does not support the conclusion that chromosome congression defects can drive tumor formation in fly models [bib_ref] Chromosome Missegregation Rate Predicts Whether Aneuploidy Will Promote or Suppress Tumors, Silk [/bib_ref] , while in mouse CIN models, a low level of CIN caused by mis-segregation of unaligned chromosomes could lead to tissue-dependent transformation [bib_ref] Aneuploidy Acts Both Oncogenically and as a Tumor Suppressor, Weaver [/bib_ref]. Elevated CIN rates after SAC perturbations, which are often associated with a large number of unaligned chromosomes, can prevent [bib_ref] Tumor Suppressor Roles of CENP-E and Nsl1 in Drosophila Epithelial Tissues, Clemente-Ruiz [/bib_ref] or promote [bib_ref] Chromosome Missegregation Rate Predicts Whether Aneuploidy Will Promote or Suppress Tumors, Silk [/bib_ref] [bib_ref] Clonal Selection of Stable Aneuploidies in Progenitor Cells Drives High-Prevalence Tumorigenesis, Trakala [/bib_ref] tumor formation in mouse and fly models. Thus, our current understanding of the connections between alignment or SAC defects and tumorigenesis is incomplete.
Regarding human cancers, different molecular elements involved in congression are altered at the genetic or protein level in certain cancers that often correlate with tumor grade and progression, including CENP-E overexpression or downregulation, Kif18a overexpression, Kif4a downregulation or overexpression [bib_ref] Reduced Expression of Cenp-e in Human Hepatocellular Carcinoma, Liu [/bib_ref] [bib_ref] Kinesin 18A Expression: Clinical Relevance to Colorectal Cancer Progression, Nagahara [/bib_ref] [bib_ref] Kif18A Is Involved in Human Breast Carcinogenesis, Zhang [/bib_ref] [bib_ref] Tumor Formation via Loss of a Molecular Motor Protein, Mazumdar [/bib_ref] , among others [bib_ref] Mechanisms of Chromosome Congression during Mitosis, Maiato [/bib_ref]. However, although, in principle, it is possible that deregulation of kinesin-related proteins involved in congression is directly tied with aneuploidy generation and tumor development, it could be related to other functions of these proteins or could be a secondary effect obtained through tumor evolution. Finally, more direct studies of tumor systems reported involvement of unaligned chromosomes in certain types of tumors. For example, unaligned chromosomes are common in high-grade serous ovarian carcinoma cells [bib_ref] Specific Mechanisms of Chromosomal Instability Indicate Therapeutic Sensitivities in High-Grade Serous Ovarian..., Tamura [/bib_ref] and to certain extent in colorectal cancer cells [bib_ref] Increased Microtubule Assembly Rates Influence Chromosomal Instability in Colorectal Cancer Cells, Ertych [/bib_ref]. In both of these tumors, unaligned chromosomes are associated with markedly elevated MT assembly rates tied to overreactive Aurora A, and in colorectal cancer, also to the deregulated BRCA1-Chk2 signaling axis [bib_ref] Specific Mechanisms of Chromosomal Instability Indicate Therapeutic Sensitivities in High-Grade Serous Ovarian..., Tamura [/bib_ref] [bib_ref] Increased Microtubule Assembly Rates Influence Chromosomal Instability in Colorectal Cancer Cells, Ertych [/bib_ref]. More recently, it was shown that phosphorylation of BRCA2, which is often mutated in breast cancer patients, by Plk1 is important for the formation of a complex between BRCA2, BubR1, and the phosphatase PP2A. A defect in this function of BRCA2 manifests itself in chromosome misalignment and unalignment, chromosome mis-segregation, mitotic delays, and aneuploidy, eventually leading to CIN [bib_ref] Proper chromosome alignment depends on BRCA2 phosphorylation by PLK1, Ehlén [/bib_ref]. Lastly, unaligned chromosomes induced by Mad2 overexpression in the mammary glands resulted in extensive CIN, which led to the generation of abnormal cells that survived the strong selective pressure of oncogene withdrawal [bib_ref] Negative Selection and Chromosome Instability Induced by Mad2 Overexpression Delay Breast Cancer..., Rowald [/bib_ref]. In agreement with these results, CIN generated by unaligned chromosomes resulted in the increased genetic diversity of cancer cells, which could evade oncogene addiction [bib_ref] Acquisition of chromosome instability is a mechanism to evade oncogene addiction, Salgueiro [/bib_ref]. There are probably additional examples of unaligned chromosomes occurring in certain tumor types, as mis-segregations are prevalent in human tumors [bib_ref] Causes and Consequences of Aneuploidy in Cancer, Gordon [/bib_ref] , but to establish direct and clear connections between the two, it would be essential to live-image cells from different tumor types throughout mitosis at high spatiotemporal resolution.
## Conclusions and future perspectives
In conclusion, polar chromosomes show a set of broad biological behaviors that separate them phenotypically from other chromosomes in human cells. Their specific features include the requirement for kinetochore motor proteins to move poleward and to congress, passage across the large polar region, passage close to the pole where the chance of acquiring erroneous attachments is higher and where end-on attachments are actively destabilized, and reliance on precise spatial and temporal control of the lateral to end-on conversion. Such peculiarity could, in principle, make peripheral polar chromosomes more prone to late-alignment, unalignment, or merotelic attachments during mitosis, especially in tumor systems that are characterized by hyper-stable MT attachments and deficient error correction mechanisms. Therefore, conditions that elevate the number of chromosomes on this alignment route, such as the prometaphase pathway of spindle assembly that frequently operates in error-prone tumor cells, could put a large burden on cellular systems that ensure error-free mitosis, mainly SAC and error correction. As error correction is often flawed in tumors, and SAC can delay mitosis only for a limited time, more erroneous attachments can persist into anaphase in cells when under a heavy burden, thereby fueling error-prone mitosis. However, much more work is needed to establish clear connections between the observed regularities of spatial pathways operating during prometaphase for different chromosomes and their tendencies to mis-segregate. In addition, complex molecular signaling mechanisms that operate during congression, error correction, and implications of those on the formation and correction of different erroneous attachments await more comprehensive studies. An important part of such studies would be long-term tracking of chromosome fates during whole mitosis in different systems, including nontransformed and tumor cells, which would lead toward achieving the goal of discriminating the cellular consequences of individual whole chromosome aneuploidies from different sources. Finally, it will be important to establish whether defects in chromosome alignment and the underlying molecular mechanisms are directly responsible for human diseases such as cancer and whether targeting chromosome congression represents an effective therapeutic approach. However, such a massive effort would require a substantial improvement in cooperation between cell biologists studying the mechanisms of mitosis and cancer researchers working on the mechanisms of tumorigenesis.
[fig] Figure 2: Mechanisms of kinetochore search-and-capture and gathering on the spindle. (A) ( [/fig]
[fig] Figure 3: Main features and models of CENP-E-mediated chromosome congression to the spindle midplane. (A) ( [/fig]
[fig] Figure 4: Mechanisms that set the distance between the chromosome and the spindle pole in bipolar and monopolar spindles. (A) Chromosome proximity to the pole where the Aurora A gradient is centered (purple gradient) controls its end-on attachment status to microtubules by the balance of poleward dynein (green arrows), anti-poleward polar ejection (PEF), and CENP-E (orange arrows) forces in the bipolar spindle. The extent of CENP-E motor activity behind and in the front of the spindle pole is controlled by the tubulin tyrosination state (microtubules in two different shades of blue). PEF activity is represented by the plus end directed walking of Kid and Kif4a motors on microtubules (red arrows) or direct microtubule pushing into the chromosome arm by polymerization (dotted lines from a microtubule tip). The thickness of the colored arrows indicates the strength of each force. MTs, microtubules. (B) Mechanisms that regulate the proximity of chromosomes to poles in the monopolar spindle. Legend as in (A). [/fig]
[fig] Cells 2022 ,: 11, x FOR PEER REVIEW 11 of 29 [/fig]
[fig] Figure 5: Models by which the degree of centrosome separation upon nuclear envelope breakdown (NEBD) influences the fidelity of chromosome segregation. (A) Two spindle poles separate before NEBD, termed the prophase pathway. This pathway is not characterized by chromosome mis-segregation. (B) Spindle poles separate after NEBD, termed the prometaphase pathway. The prometaphase pathway is associated with a three-fold increase in the number of lagging chromosomes compared to the prophase pathway through different mechanisms that promote erroneous merotelic attachments (1)-(3). An erroneous microtubule from the distal pole connected to a merotelic kinetochore is labeled red. The arrows indicate changes over time. Kinetochores with different attachment types are color labeled according to the legend on the bottom right. MT, microtubule; m, merotelic attachment. [/fig]
[fig] Figure 6: Mechanisms that control the lateral to end-on transition and error correction in different spindle regions. Different attachment types from the scheme at the top left are enlarged in panels (1)-(4). ( [/fig]
[fig] Figure 7: Routes to chromosome mis-segregation through unaligned polar chromosomes and their consequences. A process flow diagram depicting different pathways of the pseudo-metaphase cell with an unaligned polar kinetochore pair and their consequences: (1) mis-segregation of the unaligned chromosome by precocious anaphase start, (2) alignment to the metaphase plate (white area) with the merotelic attachment, which can be (left path) either resolved by error correction and result only in lazy kinetochore with appropriate attachment or (right path) left unresolved resulting in the persistent lagging chromosome with merotelic attachment, and (3) alignment of the polar kinetochore to the metaphase plate which can result in either successful biorientation (bottom path) or [/fig]
[fig] Author: Contributions: Conceptualization and original draft preparation, K.V.; review, editing, discussions, and suggestions, K.V. and I.M.T. All authors have read and agreed to the published version of the manuscript. Funding: Tolić lab is funded by the European Research Council (ERC Synergy Grant, GA Number 855158), the Croatian Science Foundation (HRZZ, project PZS-2019-02-7653), and projects co-financed by the Croatian Government and European Union through the European Regional Development Fund-the Competitiveness and Cohesion Operational Programme: IPSted (Grant KK.01.1.1.04.0057) and QuantiXLie Center of Excellence (grant KK.01.1.1.01.0004). Institutional Review Board Statement: Not applicable. Data Availability Statement: Not applicable. [/fig]
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Non-Celiac Gluten Sensitivity: The New Frontier of Gluten Related Disorders
Non Celiac Gluten sensitivity (NCGS) was originally described in the 1980s and recently a "re-discovered" disorder characterized by intestinal and extra-intestinal symptoms related to the ingestion of gluten-containing food, in subjects that are not affected with either celiac disease (CD) or wheat allergy (WA). Although NCGS frequency is still unclear, epidemiological data have been generated that can help establishing the magnitude of the problem. Clinical studies further defined the identity of NCGS and its implications in human disease. An overlap between the irritable bowel syndrome (IBS) and NCGS has been detected, requiring even more stringent diagnostic criteria. Several studies suggested a relationship between NCGS and neuropsychiatric disorders, particularly autism and schizophrenia. The first case reports of NCGS in children have been described.Nutrients 2013, 5 3841Lack of biomarkers is still a major limitation of clinical studies, making it difficult to differentiate NCGS from other gluten related disorders. Recent studies raised the possibility that, beside gluten, wheat amylase-trypsin inhibitors and low-fermentable, poorly-absorbed, short-chain carbohydrates can contribute to symptoms (at least those related to IBS) experienced by NCGS patients. In this paper we report the major advances and current trends on NCGS.
# Introduction
Gluten sensitivity (GS) was originally described in the 1980s [bib_ref] Gluten-sensitive diarrhea without evidence of celiac disease, Cooper [/bib_ref] and a recently "re-discovered" syndrome entity, characterized by intestinal and extra-intestinal symptoms related to the ingestion of gluten-containing food, in subjects that are not affected with either celiac disease (CD) or wheat allergy (WA). Following the landmark work by Sapone and coworkers, describing the clinical and diagnostic features of GS in the year 2010 [bib_ref] Differential mucosal IL-17 expression in two gliadin-induced disorders: Gluten sensitivity and the..., Sapone [/bib_ref] , a rapidly increasing number of papers have been published by many independent groups, confirming that GS should definitely be included in the spectrum of gluten-related disorders. However, many aspects of GS epidemiology, pathophysiology, clinical spectrum, and treatment are still unclear. Given the recent increase of the gluten-free market worldwide, partially sustained by individuals who claim a medical necessity to undertake a gluten-free diet (GFD), there is a need of "separating the wheat from the chaff" [bib_ref] Non celiac wheat sensitivity: Separating the wheat from the chaff, Sanders [/bib_ref]. This goal will be achieved by (a) proper scientific information, (b) shared definitions, and (c) prospective, multi-center studies addressing the many unsolved issues on GS. In order to develop a consensus on new nomenclature and classification of gluten-related disorders, a panel of experts first met in London, in February 2011. The panel proposed a series of definitions and developed a diagnostic algorithm that has been recently published [bib_ref] Spectrum of gluten-related disorders: Consensus on new nomenclature and classification, Sapone [/bib_ref].
After the 2011 London Meeting, many new papers have been published on GS. Although its frequency in the general population is still unclear, epidemiological data have been generated that can help establish the magnitude of the problem. Clinical studies further defined the identity of GS and its possible implications in human disease. An overlap between the irritable bowel syndrome (IBS) and GS has been suspected, requiring even more stringent diagnostic criteria. The first case reports of GS in children have been described. Lack of biomarkers is still a major limitation of clinical studies, making the differential diagnosis with other gluten related disorders, as well conditions independent to gluten exposure, difficult.
Evaluation and discussion of this new information was the aim of a Second Expert Meeting on GS that was held in Munich, November 30-December 2, 2012. In this paper we report the major advances and current trends on GS, as presented and debated at the Munich meeting.
## Nomenclature
At least three papers have recently addressed the issue of defining gluten-related disorders [bib_ref] Spectrum of gluten-related disorders: Consensus on new nomenclature and classification, Sapone [/bib_ref] [bib_ref] The Oslo definitions for coeliac disease and related terms, Ludvigsson [/bib_ref] [bib_ref] Lack of consensus regarding definitions of coeliac disease, Mäki [/bib_ref]. Interestingly, one of these [bib_ref] Spectrum of gluten-related disorders: Consensus on new nomenclature and classification, Sapone [/bib_ref] ranks among the most frequently downloaded paper of the publishing journal (BMC Medicine), particularly by physicians, internists or general pediatricians, and directors of diagnostic labs. There is a general agreement that the term "gluten-related disorders" is the umbrella-term to be used for describing all conditions related to ingestion of gluten-containing food. CD is a chronic small intestinal, immune-mediated, enteropathy precipitated by exposure to dietary gluten and related prolamines in genetically predisposed individuals, characterized by specific autoantibodies against tissue transglutaminase 2 (anti-TG2) and endomysium (EMA). WA is an adverse immunologic reaction to wheat proteins. In the pathogenesis of WA, wheat specific IgE antibodies play a central role, however non-IgE-mediated WA does exist [bib_ref] European Society of Pediatric Gastroenterology, Hepatology, and Nutrition. Diagnostic approach and management..., Koletzko [/bib_ref] , and this form may be difficult to distinguish from GS.
GS, which this review will focus on primarily, is a condition in which symptoms are triggered by gluten ingestion, in the absence of celiac-specific antibodies and of classical celiac villous atrophy, with variable Human Leukocyte Antigen (HLA) status and variable presence of first generation anti-gliadin antibodies (AGA). The "labeling" of this disorder was a matter of debate among the panel experts. In order to avoid confusion with CD, sometimes defined as gluten-sensitive enteropathy, "non celiac gluten sensitivity" (NCGS) appeared as an improved definition. Doubtless this is still too vague a terminology, simply reflecting the poor knowledge of the pathophysiology of this condition. As triggering cereal proteins could include fractions other than gluten (see Section 10 below) some panelists were in favor of "non-celiac wheat (protein) sensitivity", a terminology that would however conflict with the possibility that other gluten-containing cereals (rye, barley) may be offensive for the "gluten sensitive" patient. Bearing these limitations in mind, the experts' panel agreed that this entity can provisionally be defined as NCGS, a definition requiring refinement in the future.
## Epidemiology
The overall prevalence of NCGS in the general population is still unknown, mainly because many patients are currently self-diagnosed and start a GFD without medical advice or consultation. However, new data confirm that this is not an uncommon disorder at all. In a region of New Zealand, 5% of children reported non-CD-related avoidance of gluten-containing food [bib_ref] New Zealand Asthma and Allergy Cohort Study Group. Coeliac disease and gluten..., Tanpowpong [/bib_ref]. Gluten avoidance was associated with improvement of nonspecific behavioral and gastrointestinal complaints [bib_ref] Predictors of gluten avoidance and implementation of a gluten-free diet in children..., Tanpowpong [/bib_ref]. It remains to be elucidated how many children reporting gluten avoidance were indeed affected by NCGS, as the vast majority of the children involved in this study were not tested for CD nor underwent to an intestinal biopsy. In a US study performed on 7762 unselected persons aged six years or older who participated in the National Health and Nutrition Examination Survey (NHANES) 2009-2010, Digiacomo et al. found a 0.55% prevalence of persons on a self-reported GFD. The prevalence was higher in females and older participants [bib_ref] Prevalence of gluten-free diet adherence among individuals without celiac disease in the..., Digiacomo [/bib_ref]. Many of the NHANES subjects on a GFD could indeed be affected by NCGS, however this is likely to be an underestimate as (a) the possible relationship between gastro-intestinal symptoms and gluten intake was not systematically explored in this population sample, and (b) the NHANES survey was conducted before NCGS was described in the medical literature.
The analysis of the epidemiology of IBS provides an indirect estimate of intestinal NCGS frequency. According to recent population-based surveys performed in Northern Europe, the prevalence of IBS in the general adult population is 16%-25% [bib_ref] The epidemiology of irritable bowel syndrome in Denmark. A population-based survey in..., Krosgaard [/bib_ref] [bib_ref] Prevalence, comorbidity, and risk factors for functional bowel symptoms: A population-based survey..., Breckan [/bib_ref]. In a selected (and, therefore, probably biased) series of adults with IBS, the frequency of NCGS, documented by a double-blind, placebo-controlled challenge, was 28% [bib_ref] Gluten causes gastrointestinal symptoms in subjects without celiac disease: A double-blind randomized..., Biesiekierski [/bib_ref]. In the large study performed by Carroccio et al., 276 out of 920 (30%) subjects with IBS-like symptoms, according to the Rome II criteria, suffered from wheat sensitivity or multiple food hypersensitivity, including wheat sensitivity [bib_ref] Non-celiac wheat sensitivity diagnosed by double-blind placebo-controlled challenge: Exploring a new clinical..., Carroccio [/bib_ref]. Should a consistent proportion of IBS patients be affected with NCGS, the prevalence of NCGS in the general population could well be higher than CD (1%).
Although risk factors for NCGS have not yet been identified, the disorder seems to be more common in females and in young/middle age adults. The prevalence of NCGS in children is still unknown.
## Clinical picture and natural history
NCGS is characterized by symptoms that usually occur soon after gluten ingestion, disappear with gluten withdrawal and relapse following gluten challenge, within hours or few days. The "classical" presentation of NCGS is a combination of IBS-like symptoms, including abdominal pain, bloating, bowel habit abnormalities (either diarrhea or constipation), and systemic manifestations such as "foggy mind", headache, fatigue, joint and muscle pain, leg or arm numbness, dermatitis (eczema or skin rash), depression, and anemia [bib_ref] Differential mucosal IL-17 expression in two gliadin-induced disorders: Gluten sensitivity and the..., Sapone [/bib_ref] [bib_ref] Serological tests in gluten sensitivity (non celiac gluten intolerance), Volta [/bib_ref]. When seen at the specialty clinic, many NCGS patients already report the causal relationship between the ingestion of gluten-containing food and worsening of symptoms. In children, NCGS manifests with typical gastrointestinal symptoms, such as abdominal pain and chronic diarrhea, while the extra-intestinal manifestations seem to be less frequent, the most common extra-intestinal symptom being tiredness [bib_ref] Gluten sensitivity in children: Clinical, serological, genetic and histological description of the..., Mastrototaro [/bib_ref].
During the last decade, several studies suggested a relationship between NCGS and neuropsychiatric disorders (see following paragraphs).
While it is undisputable that in some cases the positive effect of gluten withdrawal can be explained by a placebo effect, this is not the case in true NCGS. In a double-blind randomized placebo-controlled study design, Biesiekierski et al. found that IBS-like symptoms of NCGS were more frequent in the gluten-treated group (68%) than in subjects on placebo (40%) [bib_ref] Gluten causes gastrointestinal symptoms in subjects without celiac disease: A double-blind randomized..., Biesiekierski [/bib_ref]. Furthermore a recent study found no significant differences between CD and NCGS patients regarding personality traits, level of somatization, quality of life, anxiety, and depressive symptoms. The somatization level was low in both diseases. Additionally, symptom increase after a gluten challenge was not related to personality in NCGS patients [bib_ref] Absence of somatization in non-coeliac gluten sensitivity, Brottveit [/bib_ref].
No major complication of untreated NCGS has so far been described; especially autoimmune comorbidity, as observed in CD, has not been reported so far. However, natural history data on NCGS are still lacking. Therefore it is difficult to draw firm conclusions on the outcome of this condition.
## Ncgs and ibs: a complex relationship
The complex relationship between IBS and dietary proteins has been recently reviewed [bib_ref] Dietary proteins and functional gastrointestinal disorders, Boettcher [/bib_ref]. Patients with CD often report symptoms compatible with IBS persisting after treatment with the GFD.
In a recent meta-analysis the pooled prevalence of IBS-type symptoms in patients with treated CD was 38.0% (95% CI, 27.0%-50.0%). The pooled odds ratio (OR) for IBS-type symptoms was higher in patients with CD than in controls (5.60; 95% CI, 3.23-9.70). In patients who were non-adherent with a GFD, the pooled OR for IBS-like symptoms, compared with those who were strictly adherent, was 2.69 (95% CI, 0.75-9.56) [bib_ref] Prevalence of irritable bowel syndrome-type symptoms in patients with celiac disease: A..., Sainsbury [/bib_ref].
That gluten ingestion may elicit gastrointestinal symptoms in non-CD patients has recently been shown in subjects affected with the D variant (diarrhea-predominant) of IBS, by Vazquez-Roque and coworkers. Subjects on a gluten containing diet (GCD) had more bowel movements per day, particularly those with HLA-DQ2 and/or DQ8 genotypes. The GCD was associated with higher small bowel permeability. Patients on the GCD had a small decrease in expression of zonula occludens 1 in small bowel mucosa, and significant decreases in expression of zonula occludens 1, claudin-1, and occludin in rectosigmoid mucosa; again the effects of the GCD on expression were significantly greater in HLA-DQ2/8-positive patients. On the other hand, the GCD vs. the GFD had no significant effects on gastrointestinal transit or histology. It was concluded that gluten alters bowel barrier functions in patients with IBS-D, particularly in HLA-DQ2/8-positive patients. These data provided mechanistic explanations for the observation that gluten withdrawal may improve patient symptoms in IBS [bib_ref] A controlled trial of gluten-free diet in patients with irritable bowel syndrome-diarrhea:..., Vazquez-Roque [/bib_ref].
How specific the effect of gluten withdrawal from the diet of patients with IBS is, still remains to be elucidated. Besides gluten, wheat, and wheat derivatives contain other constituents that could play a role in triggering symptoms in IBS patients, e.g., amylase-trypsin inhibitors (ATIs, see below) and fructans. In a second study, Biesiekirski et al. reported on 37 patients with IBS/self-reported NCGS investigated by a double-blind crossover trial. Patients were randomly assigned to a period of reduced low-fermentable, poorly-absorbed, short-chain carbohydrates (fermentable oligo-, di-, and mono-saccharides and polyols = FODMAPs) diet and then placed on either a gluten or whey proteins challenge. In all participants, gastrointestinal complaints consistently improved during reduced FODMAP intake, but significantly worsened to a similar degree when their diets included gluten or whey proteins [bib_ref] No effects of gluten in patients with self-reported non-celiac gluten sensitivity following..., Biesiekirski [/bib_ref]. FODMAPS list includes fructans, galactans, fructose, and polyols that are contained in several foodstuffs, including wheat, vegetables, and milk derivatives. These results raise the possibility that the positive effect of the GFD in patients with IBS is an unspecific consequence of reducing FODMAPs intake, given that wheat is one of the possible sources of FODMAPs. However, it should be stressed that FODMAPs cannot be entirely and exclusively responsible for the symptoms experienced by NCGS subjects, since these patients experience a resolution of symptoms while on a GFD despite continuing to ingest FODMAPs from other sources, like legumes (a much richer source of FODMPs than wheat). Nevertheless, based on the results reported by Biesiekirski et al. is also possible that there are IBS cases entirely due to FODMAPs that, therefore, cannot be classified as affected by NCGS [bib_ref] No effects of gluten in patients with self-reported non-celiac gluten sensitivity following..., Biesiekirski [/bib_ref].
## Is autism part of the ncgs spectrum?
Autism Spectrum Disorders (ASD) are chronic behavioral conditions, with onset before three years of age. ASD are one of the fastest growing developmental disabilities in the United States. They present with a wide range of stereotyped, repetitive behaviors, social and language impairment. Function and outcome is affected not only by core deficits but also by associated behaviors such as hyperactivity, aggression, anxiety, and depression. Many studies have indicated that behavioral therapy and medication may be at least partially helpful in the management of children with ASD. Research on the effect of diet and nutrition on autism has been increasing in the past two decades, particularly on the symptoms of hyperactivity and attention. One of the most popular interventions for ASD is the gluten free casein free (GFCF) diet.
The possible effect of the GFCF in children with autism is not due to underlying CD, since an association between these two conditions has never been clearly confirmed by serological screening studies [bib_ref] Autism spectrum disorder and celiac disease: No evidence for a link, Batista [/bib_ref]. It has been hypothesized that some symptoms may be caused by opioid peptides formed from the incomplete breakdown of foods containing gluten and casein. Increased intestinal permeability, also referred to as the "leaky gut syndrome," has been suspected in ASD to be part of the chain of events that allows these peptides to cross the intestinal membrane, enter the bloodstream, and cross the blood-brain barrier, affecting the endogenous opiate system and neurotransmission within the nervous system. The resulting excess of opioids is thought to lead to behaviors noted in ASD, and the removal of these substances from the diet could determine a change in autistic behaviors [bib_ref] What is the current status of research concerning use of a gluten-free,..., Marcason [/bib_ref]. The leaky gut/autism connection has fuelled a strong debate within the scientific community, far from being settled. A recent study has reported a high percentage of abnormal intestinal permeability test (as established by the lactulose/mannitol ratio) among patients with autism (36.7%) and their relatives (21.2%) compared with normal subjects (4.8%). Patients with autism on a reported GFCF diet had significantly lower intestinal permeability test values compared with those who were on an unrestricted diet and controls [bib_ref] Alterations of the intestinal barrier in patients with autism spectrum disorders and..., De Magistris [/bib_ref]. However, the degree of correlation between abnormal intestinal permeability to sugars (lactulose and mannitol) and proteins/peptides remains to be established. It should also be pointed out that, in a pilot study, Robertson et al. did not detect any changes in intestinal permeability in a small cohort of ASD children [bib_ref] Intestinal permeability and glucagon-like peptide-2 in children with autism: A controlled pilot..., Robertson [/bib_ref]. The finding of IgG class antibodies directed against food antigens is considered indirect evidence of increased intestinal permeability. Children with autism have significantly higher levels of IgG antibody (but not IgA) to gliadin compared with healthy controls, particularly in those with gastrointestinal symptoms [bib_ref] Markers of celiac disease and gluten sensitivity in children with autism, Lau [/bib_ref]. Recent studies confirmed these findings and also reported an increase in antibodies directed to several other food allergens, including casein and whole milk [bib_ref] Antibodies against food antigens in patients with autistic spectrum disorders, De Magistris [/bib_ref].
Despite its popularity, the efficacy of the GFCF diet in improving autistic behavior remains not conclusively proven. A 2008 Cochrane review reported that only two small RCTs investigated the effect of GFCF diet in children with ASD (n = 35). There were only three significant treatment effects in favor of the diet intervention: overall autistic traits, mean difference (MD) = −5.60; social isolation, MD = −3.20 and overall ability to communicate and interact, MD = 1.70. In addition three outcomes were not different between the treatment and control group while differences for ten outcomes could not be analyzed because data were skewed. The review concluded that the evidence for efficacy of these diets is poor, and large scale, good quality randomized controlled trials are needed [bib_ref] Gluten-and casein-free diets for autistic spectrum disorder, Millward [/bib_ref].
By using a two-stage, randomized, controlled study of GFCF diet of children with ASD, Whiteley and coworkers recently reported significant group improvements in core autistic and related behaviors after eight and 12 months on diet. The results showed a less dramatic change between children having been on diet for eight and children in diet for 24 months, possibly reflective of a plateau effect [bib_ref] The ScanBrit randomised, controlled, single-blind study of a gluten-and casein-free dietary intervention..., Whiteley [/bib_ref].
The above data suggest that removing gluten from the diet may positively affect the clinical outcome in some children diagnosed with ASD, indicating that autism may be part of the spectrum of NCGS, at least in some cases. However, a word of caution is necessary to stress the fact that only a small, selected sub-group of children affected by ASD may benefit from an elimination diet. Additional investigations are required in order to identify phenotypes based on best-and non-response to dietary modifications and assess any biological correlates including anthropometry before considering a dietary intervention.
## Gluten-related disorders and schizophrenia
An association between schizophrenia and CD was noted in reports spanning back to the 1960s [bib_ref] Cereals and schizophrenia data and hypothesis, Dohan [/bib_ref]. In 1986 a double-blind gluten-free/gluten-load controlled trial of 24 patients conducted by Vlissides et al. showed changes in symptom profile of schizophrenics in response to exclusion of gluten from the diet [bib_ref] A double-blind glutenfree/gluten-load controlled trial in a secure ward population, Vlissides [/bib_ref]. On the other hand, a small blind study conducted by showed no differences in the clinical status of eight schizophrenic patients on a 5-week gluten challenge in an in-patient setting, as measured by the Brief Psychiatric Rating Scale [bib_ref] Wheat gluten challenge in schizophrenic patients, Potkin [/bib_ref]. A subsequent study by Storms et al. tested 26 schizophrenic patients on a locked ward assigned to either a gluten-free or high gluten diet. No differences were found between the groups on their performance in a battery of psychological tests [bib_ref] Effects of gluten in schizophrenics, Storms [/bib_ref]. A recent study using blood samples from the Clinical Antipsychotic Trials of Intervention Effectiveness (CATIE) found that 5.5% of the subjects with schizophrenia had a high level of anti-tTG antibodies (compared to 1.1% in the healthy control sample) and 23.1% had AGA IgG positivity compared with 3.1% in controls. Interestingly enough, a large proportion of tTG positive subjects resulted EMA negative, questioning the possibility that their tTG positivity was related to CD. Indeed, only 2% of schizophrenic patients fulfilled the CD diagnostic criteria (both anti-tTG and EMA positive), questioning the role of CD in schizophrenia [bib_ref] Prevalence of celiac disease and gluten sensitivity in the United States clinical..., Cascella [/bib_ref]. Additional studies revealed that most of the tTG positive subjects were tTG-6 positive, suggesting that these antibodies are more a biomarker of neuro-inflammation than CD [bib_ref] Increased prevalence of transglutaminase 6 antibodies in sera from schizophrenia patients, Cascella [/bib_ref]. This study indicated the existence of a specific immune response to gluten in some of these patients, probably related to NCGS. Other studies confirmed the high prevalence of antibodies to AGA among people with schizophrenia [bib_ref] Markers of gluten sensitivity and celiac disease in recent-onset psychosis and multi-episode..., Dickerson [/bib_ref] , however the exact mechanism underlying the observed improvement of symptoms in some patients with the GFD has remained elusive. Immunological mechanisms have been proposed, including the assertion that a subgroup of schizophrenics suffer from food intolerances that benefit from the adoption of a GFD. The beneficial effect of a GFD may also be achieved via circulating food-derived peptides (exorphins) exerting an influence on physiological processes in the brain (same mechanism as described in the autism paragraph). If it were true that a subset of schizophrenic patients did exhibit symptoms due to sensitivity to gluten, then not only would treatment for these individuals be easier and more efficient than neuroleptics but also their quality of life would improve.
In summary, the role of NCGS in conditions affecting the nervous system remains a highly debated and controversial topic that requires additional, well-designed studies to establish the real role of gluten as a triggering factor in these diseases.
## Laboratory evaluation
So far no specific biomarker of NCGS has been identified. Recently, Volta and colleagues reported on the pattern of CD serology found in 78 untreated patients affected with NCGS. Many patients displayed an elevated prevalence of high titer, "first-generation" IgG AGA directed against native gliadin (56.4%). The prevalence of IgG AGA detected in NCGS, although lower than that found in CD (81.2%), was much higher than other pathologic conditions such as connective tissue disorders (9%) and autoimmune liver diseases (21.5%) as well as in the general population and healthy blood donors (2%-8%). On the other hand, the prevalence of IgA AGA in NCGS patients was very low (7.7%). Noteworthy, the "best" CD markers, namely IgG deamidated gliadin peptide (DGP) antibodies, IgA tTGA, and IgA EMA, were always negative in NCGS patients, except for an isolated positivity at a very low titer for IgG DGP. The consistent negativity for IgG DGP, whose synthesis "in vivo" is an expression of the interaction between tissue transglutaminase and gliadin peptides, seems to exclude the involvement of adaptive immunity in NCGS pathogenesis. Interestingly enough, ELISA activities of IgA tTGA in NCGS patients were very low with 30% of them displaying values < 1 AU (none of them had IgA deficiency) [bib_ref] Serological tests in gluten sensitivity (non celiac gluten intolerance), Volta [/bib_ref].
The CD-predisposing HLA-DQ2 and DQ8 genotypes are found in 50% of NCGS patients, a prevalence that is lower than CD (95%) and only slightly higher than the general population (30%) [bib_ref] Spectrum of gluten-related disorders: Consensus on new nomenclature and classification, Sapone [/bib_ref].
In the work of Sapone and coworkers all subjects (11 patients with NCGS, 13 with CD, and seven controls), underwent upper duodenal endoscopy for small intestinal biopsy. Those with NCGS revealed normal to mildly inflamed mucosa (Marsh 0 to 1), while all CD patients showed partial or subtotal villous atrophy with crypt hyperplasia. As expected, CD patients had increased numbers of CD3+ IELs (>50/100 enterocytes) compared to controls, while NCGS patients had a number of CD3+ IELs intermediate between CD patients and controls in the context of relatively conserved villus architecture. The numbers of TCR-γδ IELs were only elevated in CD subjects (>3.4/100 enterocytes), while in NCGS patients the numbers of γδ IELs were similar to those in controls [bib_ref] Differential mucosal IL-17 expression in two gliadin-induced disorders: Gluten sensitivity and the..., Sapone [/bib_ref]. Recently, activation of circulating basophils [bib_ref] Non-celiac wheat sensitivity diagnosed by double-blind placebo-controlled challenge: Exploring a new clinical..., Carroccio [/bib_ref] and increased infiltration of duodenal lamina propria with eosinophils [bib_ref] Non coeliac gluten sensitivity, Holmes [/bib_ref] have been described.
## Diagnosis
NCGS diagnosis is sometimes suspected by the patients themselves based on food withdrawal and introduction. Physicians may then concur if there has been the exclusion of other forms of gluten-induced disease (CD and WA) by appropriate serological and/or biopsy tests. Specific IgE might normalize if the patients are already on GFD and this might be a potential pitfall in diagnosis of WA The finding that symptoms disappear after gluten elimination adds weight to the diagnosis of NCGS, which is definitely proven by a double-blind (or open) oral gluten challenge performed after at least three weeks of GFD.
Based on a combination of clinical, biological, genetic and histological data, it is possible to differentiate the three gluten-related conditions (WA, CD, and NCGS), using recently published algorithms [bib_ref] Spectrum of gluten-related disorders: Consensus on new nomenclature and classification, Sapone [/bib_ref]. Since there is some degree of overlap between NCGS and other forms of wheat-exclusion responsive conditions (e.g., IBS responsive to low FODMAPs diet, non-IgE mediated WA), periodical patient reassessment (e.g., every 6-12 months), including an accurate dietary interview, is strongly recommended.
## Pathogenesis
The pathophysiology of NCGS is under scrutiny. In the study conducted by Sapone et al. [bib_ref] Differential mucosal IL-17 expression in two gliadin-induced disorders: Gluten sensitivity and the..., Sapone [/bib_ref] , NCGS subjects showed a normal intestinal permeability and claudin-1 and ZO-1 expression compared with celiac patients, and a significantly higher expression of claudin-4. In the same NCGS patients, the up-regulation of claudin-4 was associated with an increased expression of toll-like receptor-2 and a significant reduction of T-regulatory cell marker FoxP3 relative to controls and CD patients. Additionally, an increase in IELs of the classes α and β, but no increase in adaptive immunity-related gut mucosal gene expression, including interleukin (IL)-6, IL-21, and interferon-γ (IFN-γ), was detected in NCGS. These changes suggested an important role of the intestinal innate immune system in NCGS, without any involvement of the adaptive immune response. In a study aimed at exploring and comparing the early mucosal immunological events in CD and NCGS, Brottveit et al. confirmed that CD patients mounted a concomitant innate and adaptive immune response to gluten challenge. NCGS patients only showed increased IFN-γ levels after gluten challenge and increased density of intraepithelial CD3(+) T cells at baseline [bib_ref] Mucosal cytokine response after short-term gluten challenge in celiac disease and non-celiac..., Brottveit [/bib_ref]. These findings open the possibility of an adaptive component as well in the pathogenesis of NCGS.
The trigger/s of mucosal events leading to NCGS is not necessarily represented by the same array of gluten peptides responsible for CD development. Unlike the duodenal mucosa from patients with CD, upon incubation with gliadin, mucosa from patients with NCGS does not express markers of inflammation, and their basophils are not activated by gliadin [bib_ref] Gliadin does not induce mucosal inflammation or basophil activation in patients with..., Bucci [/bib_ref]. In vitro studies suggest that wheat ATIs could play a major role as triggers of the innate immune response in intestinal monocytes, macrophages and dendritic cells eventually leading to NCGS. Wheat ATIs are a family of five or more homologous low-molecular-weight proteins highly resistant to intestinal proteolysis. They are known to be the major allergen responsible for baker's asthma. ATIs engage the TLR4-MD2-CD14 complex and lead to up-regulation of maturation markers and elicit release of pro-inflammatory cytokines in cells from celiac and non-celiac patients and in celiac patients' biopsies [bib_ref] Wheat amylase trypsin inhibitors drive intestinal inflammation via activation of toll-like receptor..., Junker [/bib_ref].
## Current and future trends
The vast majority of celiac experts initially reacted with a great deal of skepticism to the concept of NCGS existence and the fact that it was a separate entity from CD. For those that witnessed the initial struggle of convincing health care professionals that CD was not confined within European boundaries this was a déjà vu. Indeed, we are now with NCGS where we probably were with CD forty years ago. In the 1980s we knew that CD existed, but we had little information on the mechanisms leading to the enteropathy, the genetic component of the disease, what kind of immune response was involved in the pathogenesis of the disease, its multifaceted clinical presentation, and its complication. We lacked robust screening tools to conduct well-design epidemiological studies and had little understanding on the most appropriate management of the disease and its complications. The confusion about NCGS stems from the few facts, and the many fantasies, currently available on this topic. The best testimonial of this concept is the comparison of the literature published on both conditions during the past 63 years. The publications on CD doubled every 20 years from approximately 2500 in the period of 1950-70 to ~9500 in the period 1991-2010, with already more than 2000 papers published between 2011 and 2013. Conversely, there were almost no scientific reports on NCGS before 1970 and only a handful number of papers have been published ever since, most of them after 2005. The increase interest in NCGS is testified by the decreased NCGS/CD publication ratio that dropped from 1:438 in the period 1950-70 to 1:10 in the period 2010-13 . . Trends in publication on celiac disease (CD) and non-celiac gluten sensitivity (NCGS) during the last decades. Given the limited literature on the topic, it should not come as a surprise that there are still numerous questions about NCGS that should be addressed. Is NCGS permanent or transitory? Is the threshold of sensitivity the same for everybody, or change from subject to subject and in the same subject over time? How frequent is NCGS? The range reported in the literature is between 0.5% and 6%, based on poorly conducted studies and on definitions of the disease that varies widely from one report to another. Only recently, well-conducted studies based on double blind, placebo control design are providing evidence-based data on the prevalence of NCGS in specific clinical conditions, particularly IBS [bib_ref] Gluten causes gastrointestinal symptoms in subjects without celiac disease: A double-blind randomized..., Biesiekierski [/bib_ref]. There is the strong need for more coordinated efforts to perform large multicenter studies for those conditions, including autism and schizophrenia, in which NCGS has been indicated as a possible cause in a subgroup of these patients. The lack of validated biomarkers for a diagnosis not based on exclusion criteria is judged to be of paramount importance by many experts in the field. Currently a large multicenter placebo-controlled study is underway to achieve this goal and, hopefully, will provide tools for a more correct diagnosis and for more rigorous studies to establish the prevalence of NCGS in specific conditions and in the general population. Recent studies raised the possibility that, beside gluten [bib_ref] Gluten causes gastrointestinal symptoms in subjects without celiac disease: A double-blind randomized..., Biesiekierski [/bib_ref] and wheat ATIs [bib_ref] Wheat amylase trypsin inhibitors drive intestinal inflammation via activation of toll-like receptor..., Junker [/bib_ref] , low-fermentable, poorly-absorbed, short-chain carbohydrates [bib_ref] No effects of gluten in patients with self-reported non-celiac gluten sensitivity following..., Biesiekirski [/bib_ref] can contribute to symptoms (at least those related to IBS) experienced by NCGS patients. These new findings need corroboration through additional studies involving larger numbers of subjects. If these studies will confirm these new findings, they will probably prompt a change in nomenclature from NCGS to wheat sensitivity to reflect the fact that, beside gluten, other components of wheat may be responsible for the symptoms reported by NCGS patients.
## Conflicts of interest
Carlo Catassi received consulting fees from Schär and Menarini Diagnostics. Luca Elli and Anna Sapone received consulting fees from Schär. Peter Green is a member of the scientific advisory board of Alvine Pharmaceuticals and Alba Therapeutics. Alessio Fasano owns stock in Alba Therapeutics. The other authors declared no conflict of interest.
© 2013 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).
AcknowledgmentsWe wish to express our gratitude to Jacqueline Pante (Schär, Merano, Italy) and Andrea Comaschi (Weber Shandwick, Milan, Italy) for logistic support in the organization of the Munich Meeting. |
Barriers to recognition of out-of-hospital cardiac arrest during emergency medical calls: a qualitative inductive thematic analysis
Background:The chance of surviving out-of-hospital cardiac arrest (OHCA) depends on early and correct recognition of cardiac arrest by the emergency medical dispatcher during the emergency call. When cardiac arrest is identified, telephone guided cardiopulmonary resuscitation (CPR) and referral to an automated external defibrillator should be initiated. Previous studies have investigated barriers to recognition of OHCA, and found the caller's description of sign of life, the type of caller, caller's emotional state, an inadequate dialogue during the emergency call, and patient's agonal breathing as influential factors. Though many of these factors are included in the algorithms used by medical dispatchers, many OHCA still remain not recognised. Qualitative studies investigating the communication between the caller and dispatcher are very scarce. There is a lack of knowledge about what influences the dispatchers' recognition of OHCA, focusing on the communication during the emergency call. The purpose of this study is to identify factors affecting medical dispatchers' recognition of OHCA during emergency calls in a qualitative analysis of calls. Methods: An investigator triangulated inductive thematic analysis of recordings of out-of-hospital cardiac arrest emergency calls from December 2012. Participants were the callers (bystanders) and the emergency medical dispatchers. Data were analysed using a hermeneutic approach.Results: Based on the concept of data saturation, 13 recordings of not recognised cardiac arrest and 8 recordings of recognised cardiac arrests were analysed. Three main themes, six subthemes and an embedded theme emerged from the analysis: caller's physical distance (caller near patient, caller not near patient), caller's emotional distance (keeping calm, losing control), caller is a healthcare professional (responsibility is handed over to the caller, caller assumes responsibility), and the embedded theme: caller assesses the patient. Conclusion: The physical and emotional proximity of the caller (bystander) as well as the caller's professional background affect the dispatcher's chances of correct recognition and handling of cardiac arrest. The dispatcher should acknowledge the triple roles of conducting patient assessment, instructing the caller, and reassuring the emotionally affected caller.
# Background
The chance of surviving from out-of-hospital cardiac arrest (OHCA) is highly associated with emergency medical dispatchers' recognition of the condition during emergency calls, early bystander cardiopulmonary resuscitation (CPR), and early defibrillation [bib_ref] Association of national initiatives to improve cardiac arrest management with rates of..., Wissenberg [/bib_ref] [bib_ref] Emergency medical service dispatch cardiopulmonary resuscitation prearrival instructions to improve survival from..., Lerner [/bib_ref] [bib_ref] Understanding and improving low bystander CPR rates: a systematic review of the..., Vaillancourt [/bib_ref] [bib_ref] Importance of the first link: description and recognition of an out-of-hospital cardiac..., Berdowski [/bib_ref]. It is essential that the emergency medical dispatcher (EMD) recognises OHCA, so telephone assisted CPR (tCPR) and referral to an automated external defibrillator (AED) can be initiated. Earlier studies have investigated barriers to recognition of OHCA, and found the caller's description of signs of life, the type of caller, caller's emotional state, inadequate dialogue during the emergency call, and patient's agonal breathing as influential factors. Moreover, successful recognition of OHCA is associated with an assessment of the patient's consciousness, breathing pattern, and facial colour [bib_ref] Tuition of emergency medical dispatchers in the recognition of agonal respiration increases..., Bohm [/bib_ref] [bib_ref] Dispatcher assessments for agonal breathing improve detection of cardiac arrest, Roppolo [/bib_ref] [bib_ref] Interaction between emergency medical dispatcher and caller in suspected out-of-hospital cardiac arrest..., Bang [/bib_ref] [bib_ref] In patients with out-of-hospital cardiac arrest, does the provision of dispatch cardiopulmonary..., Bohm [/bib_ref] [bib_ref] Perceptions of collapse and assessment of cardiac arrest by bystanders of out-of-hospital..., Breckwoldt [/bib_ref]. Many of these factors are included in the algorithms used by medical dispatchers, but still, not all OHCA's are recognised [bib_ref] Accuracy of determining cardiac arrest by emergency medical dispatchers, Clark [/bib_ref] [bib_ref] Does the use of the Advanced Medical Priority Dispatch System affect cardiac..., Heward [/bib_ref] [bib_ref] Do health care professionals report sudden cardiac arrest better than laymen?, Castrén [/bib_ref] [bib_ref] Emergency call processing and survival from out-of-hospital ventricular fibrillation, Kuisma [/bib_ref] [bib_ref] Emergency CPR instruction via telephone, Eisenberg [/bib_ref] [bib_ref] Evaluation of dispatcherassisted cardiopulmonary resuscitation, Bång [/bib_ref] [bib_ref] Factors impeding dispatcher-assisted telephone cardiopulmonary resuscitation, Hauff [/bib_ref] [bib_ref] Sensitivity and specificity of the medical priority dispatch system in detecting cardiac..., Flynn [/bib_ref] [bib_ref] The accuracy of predicting cardiac arrest by emergency medical services dispatchers: the..., Garza [/bib_ref] [bib_ref] The obstacles to maximising the impact of public access defibrillation: an assessment..., Cairns [/bib_ref]. A better understanding of factors leading to successful recognition of OHCA has the potential to increase the proportion of bystander CPR, and thereby improve survival rate [bib_ref] European Resuscitation Council Guidelines for Resuscitation 2010 Section 1. Executive summary, Nolan [/bib_ref]. Qualitative studies investigating recognition of OHCA, focusing on the communication between caller and dispatcher are needed. The aim of this study was to identify factors affecting medical dispatchers' recognition of OHCA during emergency calls in the Emergency Medical Services (EMS), Copenhagen using a qualitative analysis.
# Methods
## Setting
The study took place at the Emergency Medical Dispatch Center (EMDC) in Copenhagen, Denmark serving a population of 1.7 million and responding to about 110.000 emergency calls annually. In Denmark, there is a single emergency phone number (1-1-2) leading to an emergency call center. In case of a medical problem, the caller is re-directed to an EMDC that responds to the call by activating the appropriate response. The medical dispatchers are specially trained registered nurses and paramedics who are supported by a criteria-based, nationwide priority tool (Danish Index for Emergency Care). For each call, the system recommends a response according to contact cause and urgency of the condition. The algorithm is described in [fig_ref] Figure 1: Initial steps in the algorithm for dispatch assisted telephone CPR [/fig_ref] , and the response types are described in [fig_ref] Table 1: Emergency response types [/fig_ref].
In each emergency call, the dispatcher starts by asking the caller "How can I help you?" and then assesses the patient's consciousness and breathing pattern. If an OHCA is suspected, the dispatcher provides telephone assisted CPR to the bystander and refers to the nearest AED. In case of suspected OHCA, a high priority response including an emergency physician is dispatched.
## Study design
An inductive qualitative approach was chosen, using recordings of emergency calls including recognised and not recognised OHCA as the source of data. The participants were callers (bystanders) and medical dispatchers. The goal of qualitative research is to understand and describe a phenomenon, including its definition, terminology and relation to other phenomena. Detailed description of the caller-dispatcher conversation during emergency calls paves the way to a better understanding of why OHCA is or is not recognised during the emergency call. The study was conducted in two steps: 1) sorting the recordings according to recognised and not recognised OHCA, and 2) analysing the recordings and identifying factors related to recognised and unrecognised OHCA.
## Preconceptions of the investigators
An active and deliberate relationship to their preconception or potential prejudices can aid the investigators to learn from the data. In the present study, the team of investigators analysing data consisted of a registered nurse (DA), a physician (TPM), a professor in clinical nursing (IE), and the medical director of EMS Copenhagen (FKL). The specific knowledge of OHCA and EMS systems were paramount to the sorting of data items, while the difference in experience and interdisciplinary knowledge among team members facilitated critical reflection and reduced blind spots. The use of investigator triangulation strengthens the design by supplementing and challenging the investigators' statements during the process of analysis.
## Sampling strategy
The sample was collected in December 2012 (31 days) and the sampling strategy was criterion-based selection. The calls during December 2012 were chosen because they were the most recent calls available. The criterion for selection were all calls regarding sudden unexpected cardiac arrest. Cardiac arrest due to trauma, terminal disease and patients with definite signs of death (head separated from body, rigor mortis, incineration etc.) were excluded. The OHCA calls were identified by merging databases containing mobile critical care unit charts and EMDC data. OHCA was considered as recognised if CPR was encouraged, guided, or conducted at any time during the call. Sample size was determined by saturation of data, which means that inclusion of calls continued until information became redundant, and the themes appeared robust.
# Analysis
Thematic analysis as described by Braun & Clarke was used, which identifies, analyses, and describes patterns in data. The method is not bound by any specific theory, and does not put any specific demands on the data collection method. This pragmatic methodology lends itself for this study, because of the exploratory focus. The process of thematic analysis progresses through six phases: Phase 1: Familiarisation with data, phase 2: generating initial codes, phase 3: searching for themes, phase 4: reviewing themes, phase 5 defining and naming themes, and phase 6: producing the report. Analysis continued until saturation, viz. when information became redundant. Recordings were transcribed by simple orthographic method. During analysis the recordings were played as a supplement to transcribed data in order to exploit contextual nuances.
# Ethical considerations
# Results
We included 21 emergency calls in the study; 8 with recognised OHCA and 13 with not recognised OHCA. [fig_ref] Table 2: Characteristics for emergency calls concerning recognised OHCA Patient no [/fig_ref] show an identification number for each call, patient age and gender, the caller's relation to the patient, the caller's proximity to the patient and the assigned response to the emergency call for recognized and not recognised OHCA, respectively.
## Themes
Three themes emerged during analysis (
## Caller's physical distance caller near patient
In all recognised OHCA calls, the caller was in close proximity to the patient. This enabled better communication and assessment, and the medical dispatcher was able to react quickly to information and initiate the EMD algorithm systematically with little or no interruption. If the caller was with a patient and able to describe abnormal breathing, OHCA was recognised at an early stage. During some calls the callers presented a patient with normal breathing, which changed the structure of the conversation. The dispatchers then abandoned the algorithm in favour of providing first aid, and thereby missed the provision of tCPR. If the caller stated later that the patient was deteriorating, the algorithm was applied again. This was evident in several calls where the caller interrupted the dispatcher, to describe the new situation. In the following quotation, the caller initially set the scene by presenting a respiratory problem. The dispatcher was able to explore the situation and apply the algorithm.
[Patient 4] Dispatcher: "Hello this is the nurse from the dispatch centre". Caller: "I'm calling from [nursing home]. I have a resident who has fallen and now he has trouble breathing; he is turning blue". Dispatcher: "He is turning blue?" Caller: "Yes". Dispatcher: "He is awake, isn't he?" Caller: "More or less; no he is not". Dispatcher: "He is not responsive?" Caller: "No, [calling name of patient]". Dispatcher: "Is he
## Caller not near patient
As soon as it was clear to the dispatcher that the caller was not present at the scene, the algorithm from the EMD was disregarded, and a response was dispatched based on the caller's description of the patient's condition. In cases where the caller was closer to the patient, but not in the same room, the dispatcher had to ask the caller to go to the patient and assess the patient. These situations seemed particularly challenging when the caller was emotionally affected by the situation. The dispatcher had a double role of calming the caller while gathering relevant information. In the following the caller is calling on behalf of his mother in another part of the country.
[Patient 13] Caller: "Yes, hello. You are speaking with [name]. My mother has just called, that her husband has collapsed. A couple of months ago he had an angioplasty". Dispatcher: "Yes". Caller: "and he has fainted and is unresponsive". Dispatcher: "I see".
Caller: "She [mother] isn't well, has seizures and …" Dispatcher: "Is it your mother or him?" Caller: "Him. My mother is so weak that she apparently isn't able to call [mother's location]. I am in [caller's location] and she lives at [mother's location]".
## Caller assesses the patient
If the caller was close to the patient and able to make an assessment, this was reported to the dispatcher. In most of the recordings, the patient's problem was initially related to respiratory failure or loss of consciousness. The dispatcher explored these avenues using the algorithm, but if the caller stated that breathing was present, the algorithm for cardiac arrest was abandoned. In some cases, the dispatcher continued to obtain a patient history without recognizing the arrest until the arrival of ambulance. The following quote illustrates a situation, where the caller believed the patient was breathing and the dispatcher as a consequence abandoned the algorithm.
[Patient 17] Dispatcher: "Okay, but is he unconscious and not reacting to pain?" Caller: "(…), well he is breathing (…), but not reacting". Dispatcher: "Then you have to make sure he is breathing, and that he hasn't arrested (…) try to see if his chest is moving." Caller: "It is." Dispatcher: "It is, and is it moving properly?" Caller: "I would think so (…)".
## Caller's emotional distance keeping calm
In calls with recognised OHCA, the dispatcher was able to provide instructions to the caller in a calm, clear and direct way, and the caller followed the instructions. The caller became an active partner in patient assessment adding to the success of the call. The dispatcher was able to establish a good communication with the caller and systematically follow the algorithm. The following is an example of direct and good communication.
[Patient 2] Caller: "Come on, again. Breathe. It's as if he's dropping his tongue, like he can't hold it himself." Dispatcher: "No, but if you hold the jaw, pulling the" (caller interrupts) Caller: "I have two fingers inside the mouth, holding the tongue." Dispatcher: "You pull the jaw forward" (speaking slowly and loud). Caller: "Come on. Hell, yes there it was" (loud and strained, then relieved). Dispatcher: "That's great, and how is his colour now, any better?" Caller: "No, he is still completely blue and completely dead" (calm and clear). Dispatcher: "Try to put him onto his back. You have to start CPR."
## Losing control
An emotionally affected caller had more trouble recognising OHCA. The dispatcher assessed the condition of the caller listening for signs of crying or talking. In not recognised OHCA the dispatcher might choose to calm the caller before continuing. In some cases, the dispatcher became emotionally affected in response to the caller's distress. In the following, the dispatcher tried to maintain patient assessment despite the distress of the caller.
[Patient 20] Dispatcher: "You need to pay attention; I'm going to ask you some questions. I already sent an ambulance, okay?" Caller: "Yes. (…)." Dispatcher: "Is he breathing, your husband? " Caller: "Yes, I hope (trembling voice)". Dispatcher: "What is the colour of his face?" (Interrupting) Caller: "Well I can't tell you more, now I have to go. You have to come (loud and Caller is a healthcare professional Responsibility is handed over to the caller When the dispatcher recognised the caller as a healthcare professional, the roles were reversed; the caller was given responsibility and took action while the dispatcher had a counselling role. In recordings with recognised OHCA, the caller recognized the warning signs. In the following the caller identified OHCA and the dispatcher initiated tCPR.
[Patient 8] Dispatcher: "Uhm, does he have any heart disease or pulmonary diseases?" Caller: "Yes, COPD and something with his heart". Dispatcher: "Okay, it can be either one or the other we are dealing with." Caller: "Yes, I just came here, because, uhm, I had to check his medications and will initiate a resuscitation attempt because he stopped breathing (…)." Dispatcher: "Do you have a defibrillator?" Caller: "No (…)." Dispatcher: "How's the breathing now?" Caller: "Right now he is pausing and the pauses are getting longer". Dispatcher: "How about the chest, does it move up and down when he breathes?" Caller: "Uhm, right now there is no breathing." Dispatcher: "Then you might want to start CPR".
## Caller assumes responsibility
In some calls with not recognised OHCA, the dispatcher indirectly transferred the responsibility to the caller, if a healthcare professional. This phenomenon was characterised by a relaxed collegial atmosphere, where the algorithm for cardiac arrest was abandoned in favour of more informal assessment and the use of ambiguous terms, e.g. "fairly unconscious".
[Patient 21] Caller: "He is fairly unconscious at the moment (…)." Dispatcher: "Is he breathing now?" Caller: "He is breathing now and then, and then he stops, uhm, it sound like fluids are accumulating (…)". Dispatcher: "Okay, and now you are telling me that he is unconscious?" Caller: "He is unconscious now, yes." Dispatcher: "Yes, okay. And breathing is slower…" Caller: "Slower breathing (…)" Dispatcher: "Good, then that's settled, bye."
# Discussion
The aim of this study was to identify factors affecting medical dispatchers' recognition of OHCA during emergency calls. The main findings included three themes: "caller's physical distance", "caller's emotional distance", and "caller is a healthcare professional". The dispatchers often had a triple role; trying to obtain patient information, instructing the caller, and calming the caller. This triple role was evident in two subthemes: "Caller not near the patient" and "losing control". In our study, the dispatcher did not strictly use the cardiac arrest algorithm if the caller was not near the patient. This finding is supported by several studies that have demonstrated the distance between caller and patient being a potential barrier to the recognition of OHCA [bib_ref] Factors impeding dispatcher-assisted telephone cardiopulmonary resuscitation, Hauff [/bib_ref] [bib_ref] Dispatcher-assisted telephone CPR: Common delays and time standards for delivery, Culley [/bib_ref] [bib_ref] Out-of-hospital cardiac arrests in Helsinki: Utstein style reporting, Kuisma [/bib_ref] [bib_ref] Identification of cardiac arrest by emergency dispatchers, Eisenberg [/bib_ref]. One study including interviews with 10 dispatchers showed that tCPR was unlikely if the caller was not at the scene [bib_ref] Dispatcher-assisted telephone CPR: a qualitative study exploring how dispatchers perceive their experiences, Bång [/bib_ref]. The same study showed that the emotional state of the caller influenced initiation of tCPR. In accordance with our study, several studies have shown how the emotional state of the caller affected OHCA recognition and precluded tCPR [bib_ref] Identification of cardiac arrest by emergency dispatchers, Eisenberg [/bib_ref] [bib_ref] The emotional content and cooperation score in emergency medical dispatching, Clawson [/bib_ref] [bib_ref] When the unreal becomes real: family members' experiences of cardiac arrest, Weslien [/bib_ref] [bib_ref] Do CPR-trained Bystanders Perform CPR?, Swor [/bib_ref].
Our study has shown that it is risky if the dispatcher disregards the algorithm, even if the situation appears under control. Similar results have been reported in a recent study showing that the main barriers to recognition of OHCA were absence or incomplete assessment of breathing and the presence of agonal breathing [bib_ref] Out-of-hospital cardiac arrest phone detection: Those who most need chest compressions are..., Travers [/bib_ref].
In our sub-theme "losing control" we found that the emotional state of the caller could influence the dispatcher. We also observed the inherent emotional asymmetry between caller and dispatcher, as the caller was typically experiencing a unique and devastating situation, whereas the dispatcher was doing "business as usual". In the sub-theme "losing control" the asymmetry was evident, but in some cases the dispatchers also got close to losing control. If, on the other hand, the caller was calm, or reassured by the dispatcher, the asymmetry was replaced by a constructive and information rich conversation.
The characteristics of the callers, as illustrated in Tables 2 and 3, showed that all callers in the group that recognised OHCA were in close proximity to the patient. This was supported in the theme "caller's physical distance". Importantly, more patients were successfully resuscitated when OHCA was recognised, potentially increasing patients' chances of survival [bib_ref] European Resuscitation Council Guidelines for Resuscitation 2010 Section 1. Executive summary, Nolan [/bib_ref].
Surprisingly, the theme "caller is a healthcare professional", showed that the caller's profession did not necessarily increase the chances of OHCA recognition. Some of these callers were nursing home staff, and might not be as updated in emergency care and cardiac arrest, as presumed by the dispatcher. When the dispatcher let the caller take command and control, the cardiac arrest algorithm was abandoned with poor results. Dispatchers' use of the algorithm is a modifiable factor, and it should be emphasized that the algorithm must be used even if the caller is a healthcare professional. Other studies have described the phenomenon of significantly lower OHCA recognition in professionals than non-professional bystanders, and less use of the algorithm among healthcare professionals [bib_ref] Do health care professionals report sudden cardiac arrest better than laymen?, Castrén [/bib_ref].
In our embedded theme "caller assesses the patient" we found that the dispatcher abandoned the cardiac arrest algorithm if the caller identified breathing or signs of life. Other studies have found that the description of signs of life is one of the most significant factors leading to failed recognition of OHCA [bib_ref] Factors impeding dispatcher-assisted telephone cardiopulmonary resuscitation, Hauff [/bib_ref]. Reasons for this might be that the patient does not have cardiac arrest before the arrival of the ambulance, or especially, that the patient has agonal breathing, which is present in 40 % of OHCA's, and is known to delay or impede recognition of OHCA [bib_ref] Factors impeding dispatcher-assisted telephone cardiopulmonary resuscitation, Hauff [/bib_ref] [bib_ref] Dispatcher-assisted telephone CPR: a qualitative study exploring how dispatchers perceive their experiences, Bång [/bib_ref] [bib_ref] Incidence and significance of gasping or agonal respirations in cardiac arrest patients, Eisenberg [/bib_ref] [bib_ref] Incidence of agonal respirations in sudden cardiac arrest, Clark [/bib_ref] [bib_ref] Agonal respirations during cardiac arrest, Rea [/bib_ref] [bib_ref] Dispatch-assisted cardiopulmonary resuscitation: the anchor link in the chain of survival, Bobrow [/bib_ref]. Another reason could be bystanders' difficulties in assessing breathing and consciousness, potentially leading to incorrect information passed to the dispatcher [bib_ref] Debriefing bystanders of out-of-hospital cardiac arrest is valuable, Møller [/bib_ref]. An implication of this knowledge could be to acknowledge the dispatcher, caller and other bystanders as a "first resuscitation team" working towards the common goal of recognising OHCA [bib_ref] Challenges in out-of-hospital cardiac arrest -a study combining closed-circuit television (CCTV) and..., Linderoth [/bib_ref].
Inductive thematic analysis has been a strong methodological tool to meet the purpose of this study. There are limitations, which should be addressed. Most importantly, we cannot be sure if calls identified as not recognised OHCA, occurred prior to the call or after termination of the call before arrival of the ambulance. It can be argued, however, that OHCA was imminent and therefore still essential to recognise. To support the credibility of the study, investigator triangulation was used. Generalizability was increased by external studies showing similar results and by other dispatchers in Europe using the same EMD system. Reliability was achieved by saturation of data.
# Conclusion
The study shows that the caller has a central role in recognising and acting upon OHCA during emergency calls. The physical and emotional proximity of the caller (bystander) as well as the caller's professional background affect the dispatcher's chances of correct recognition and handling of cardiac arrest. The dispatcher should acknowledge the triple roles of conducting patient assessment, instructing the caller, and reassuring the emotionally affected caller.
[fig] Figure 1: Initial steps in the algorithm for dispatch assisted telephone CPR [/fig]
[table] Table 4: : caller's physical distance (caller near patient, caller not near [/table]
[table] Table 1: Emergency response types [/table]
[table] Table 2: Characteristics for emergency calls concerning recognised OHCA Patient no. Patient age Patient gender Caller's relation to patient Caller' s proximity to patient Response Emergency physician treatment [/table]
[table] Table 3: Characteristics for emergency calls concerning not recognised OHCA Patient no. Patient age Patient gender Caller's relation to patient Caller's distance to patient Response Emergency physician treatment Near patient: The caller cannot see the patient, but communicate with a person together with the patient. Not at scene: Caller in different location from the patient. Resuscitation success: CPR successful. Resuscitation failed: CPR attempted, but unsuccessful. No treatment: CPR not attempted. a Indicates healthcare professional. A1 = Mobile Critical Care Unit and ambulance staffed with paramedic, A2 = Ambulance staffed with a paramedic, A3 = Basic life support ambulance, B2 = Ambulance staffed with a paramedic, no lights and siren breathing?" Caller: "Well … [pause]". Dispatcher: "I guess not. Then you should start CPR". [/table]
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Delta wave automatic mapping and catheter ablation without fluoroscopy in patients with overt accessory pathway: A new workflow
# Introduction
Accessory pathways (AP) are abnormal electrical connections between 2 parts of the heart, usually isolated one from the other. Atrioventricular AP may be responsible for atrioventricular reentrant tachycardia (AVRT), which accounts for about 30% of supraventricular tachycardias, being especially common in the pediatric population. [bib_ref] Supraventricular tachycardia, Delacretaz [/bib_ref] [bib_ref] Supraventricular tachycardia: diagnosis and management, Fox [/bib_ref] When an AP is visible on a resting electrocardiogram (ECG) (ie, overt or manifest AP), it results in the so-called pre-excitation, which leads to Wolff-Parkinson-White syndrome when associated to tachyarrhythmias.
According to current guidelines (ESC 2019), [bib_ref] ESC Guidelines for the management of patients with supraventricular tachycardia. The task..., Brugada [/bib_ref] catheter ablation of AP is recommended in patients with symptomatic and recurrent AVRT, or in asymptomatic patients with highrisk profile for specific electrophysiological (EP) AP properties (shortest pre-excited R-R interval 250 ms, AP effective refractory period 250 ms, multiple APs, and an inducible AP-mediated tachycardia), or for certain patients' characteristics (dangerous occupation, competitive athletes).
In the last years, electroanatomical (EA) mapping systems have represented an alternative and an assistance to conventional EP study, permitting the reduction of fluoroscopy time and radiation dose.
Traditionally, before EA mapping, there have been multiple key factors for successful ablation of an AP: catheter technology, catheter stability, and careful analysis of the signals and intracardiac electrograms (EGM) measured by the distal electrodes of the ablation probe, as well as an essential element represented by the operator experience (particularly in recognizing a signal that leads to AP disruption if ablated). [bib_ref] Novel method of signal analysis for ablation of Wolff-Parkinson-White syndrome, Ceresnak [/bib_ref] Additional parameters are used for correct localization of AP, such as measurement of local AV time and time from local ventricular electrogram and delta wave at the surface ECG. Nonetheless, these parameters can also be inaccurate, especially if used individually.
In this context, there is still a significant failure rate of AP ablation, with an average recurrence rate of 8%, which can increase up to 17% in particular cases such as septal AP. [bib_ref] ACC/AHA/HRS Guideline for the Management of Adult Patients With Supraventricular Tachycardia, Page [/bib_ref] With the aim to improve diagnosis and treatment success of AP, we developed a new workflow based on EA mapping using the CARTOÒ 3 System (Biosense Webster, Johnson & Johnson Medical S.p.a., Irvine, CA).
## Key teaching points
Traditionally, careful analysis of the signals and intracardiac electrograms is required to address accessory pathways (AP). To correctly localize an overt AP, measurements of local AV time and time from local ventricular electrogram and delta wave at the surface electrocardiogram are required. These parameters can be inaccurate and manual annotation during electroanatomical mapping may lead to errors.
With the CARTO 3 System (Biosense Webster) and an automatic mapping workflow that combines the Confidense module (Automatic Mapping, Wavefront Annotation, and Pattern Matching) and the CARTO Prime Parallel Mapping module, it is possible to precisely localize the AP, optimizing the radiofrequency applications and achieving a safe and successful endpoint, as demonstrated by the 3 cases presented.
The proposed automatic workflow enables fast mapping and avoids inaccuracies that may occur during manual annotations of the signals. This workflow consists of a multiparametric evaluation and an automatic mapping process to localize the AP. The purpose is to achieve a fast and precise localization of the AP minimizing the radiofrequency (RF) applications and the total RF time, and eventually increasing the success rate of the ablation. Nevertheless, as already demonstrated in the literature, [bib_ref] Predictors of zero X-ray ablation for supraventricular tachycardias in a nationwide multicenter..., Pani [/bib_ref] using an EA mapping system enables to minimize or even eliminate fluoroscopy.
## Workflow description
The workflow is based on the concomitant use of the following modules integrated in the CARTO PrimeÔ software of the CARTO 3 system version 7 (Biosense Webster): ConfidenseÔ module (Automatic Mapping, Wavefront Annotation, and Pattern Matching) and CARTO PrimeÔ Parallel Mapping module. For EA mapping and arrhythmic substrate ablation, we used the ThermoCool SmartTouchÒ catheter (Biosense Webster, Inc), an irrigated catheter with contact force sensing and vector visualization.
By relying on this workflow, for some patients (when affected by right-sided manifest AP), it was possible to use only a ThermoCool SmartTouch catheter without employing other intracavitary references.
First, we acquired the basal ECG signal for Pattern Matching (beat-to-beat analysis on the 12-lead ECG derivation), setting the reference on the most stable QRS peak in 1 of the 12 ECG leads. This reference lead was expected not to change polarity after ablation.
Then, using the Parallel Mapping module, we acquired simultaneously 2 different EA maps. The first map (a bipolar map with 0.05-0.5 mV standard cut-off for atrial mapping) was aimed to the atrial signal, in order to define the precise localization of the annular plane and other anatomical structures (coronary sinus ostium, inferior and superior vena cava, etc). For this map the window of interest (WOI) was set excluding ventricular activity (WOI: -250/-50; reference: QRS peak). In the second map, information on the local activation time of the overt AP was acquired, excluding the atrial signals from the WOI; in this way, we always had only 1 signal in the reference window. Specifically, the WOI started at the end of the P wave, evaluated on every lead in sinus rhythm.
The annotation of the ventricular signal in the WOI is based on the Wavefront Annotation algorithm of the CARTO 3 system. This algorithm focuses on the automatic annotation of the maximum negative slope of the unipolar EGM within the window of interest defined on the bipolar EGM. All the signals were annotated automatically by the algorithm, without any manual adjustment.
In the present report, we describe 3 clinical cases of overt AP ablation using this new workflow.
## Case report case 1
A 31-year-old woman presented to outpatient visit for frequent episodes of undocumented palpitations. The 12lead ECG showed a ventricular pre-excitation, and the EP study revealed the presence of a right posteroseptal AP, with easy induction of AVRT during ventricular stimulation (tachycardia cycle length 5 280 ms). We mapped the right atrium with a ThermoCool SmartTouch catheter [fig_ref] Figure 1: Case 1 [/fig_ref] and placed a decapolar and a tetrapolar catheter in the coronary sinus and in the right ventricle, respectively. We applied the proposed workflow, achieving a localization of the AP on the 3-D map, confirmed by the maximum AV fusion and the best QS signal on the unipolar derivation. After 1.2 seconds of RF delivery at 35 W with a contact force .10 g, AP disappearance was observed (time-to-effect 5 4 seconds). No fluoroscopy was required during the entire procedure.
## Case 2
The second case involves a 17-year-old male patient. Delta waves were occasionally observed on the ECG during a sport medical visit. The patient was asymptomatic for palpitations or tachycardia. Nevertheless, being a competitive athlete, he underwent an EP study. An EA mapping of the right chambers and left atrium was created using only 1 mapping/ablation catheter (ThermoCool SmartTouch 3.5 mm D-F irrigated catheter, Biosense Webster). The left atrium was reached via patent foramen ovale (PFO). A decapolar catheter was placed in the coronary sinus, whereas 2 tetrapolar catheters were placed, 1 in the right ventricle and 1 in the right atrium, respectively. The EP study confirmed the presence of a left posterior AP with high-risk properties (ERP of 250 ms).
Therefore, using the previously described workflow, we obtained a precise localization of the AP [fig_ref] Figure 2: Case 2 [/fig_ref]. After finding the maximum AV fusion and the best QS signal on the unipolar lead (which confirmed the localization), RF was delivered with a power of 35 W and a contact force .10 g, leading to the immediate disappearance of the pre-excitation (time-to-effect ,2 seconds). No fluoroscopy was required for catheter placement or during ablation.
## Case 3
A 53-year-old woman, with a known diagnosis of ECG preexcitation, symptomatic for dizziness and palpitations, underwent an EP study. The examination revealed the presence of an anterolateral left manifest AP, with an ERP of 290 ms, and induction of AVRT. A decapolar and a tetrapolar catheter were placed in the coronary sinus and in the right ventricle, respectively, whereas the ThermoCool SmartTouch catheter was used in the left ventricle for activation mapping. By applying the proposed workflow, we identified the location of the AP, which was confirmed by the maximum AV fusion and the best QS signal on the unipolar derivation [fig_ref] Figure 3: Case 3 [/fig_ref]. After 2 seconds of RF delivery at 35 W and with a contact force .10 g, AP disappearance was observed (time-to-effect 5 2 seconds).
Fluoroscopy was only limited to catheter placement (especially for transaortic approach), with a total fluoroscopy time of 30 seconds and an effective dose of 0.07 mSv.
# Discussion and conclusions
As shown in the 3 cases reported, the proposed workflow allowed a fast and precise annotation of the signal of interest, increasing the precision of the EA mapping. A short timeto-effect (defined as AP disappearing) during ablation was also achieved, optimizing intraprocedural outcome. Specifically, we observed a time-to-effect 5 seconds. Nevertheless, an ablation index of 450 was always reached. Mean procedural time was 70 minutes, including 30 minutes of waiting time. For a map of approximately 200 points, the average mapping time was 5 minutes. When mapping was restricted to a smaller area with fewer acquired points, then the mapping time was even shorter.
In our experience, using this workflow, the mapping time was reduced by 5-10 minutes compared to the conventional approach.
For all the patients Holter ECG (7 days) was performed at 1 and 6 months after the procedure and no pre-excitation was observed, nor have symptoms of SVT been reported.
By using the pattern matching module, we do not have to rely on any intracavitary reference for the activation map and we can map using solely the SmartTouch catheter. Furthermore, no additional multielectrode catheters such as PentaRayÒ or DecaNavÒ were required to achieve successful endpoint, hence minimizing the number of catheters employed and the risk of complications.
The advantage of this workflow is that it excludes the first atrial signal and consistently annotates the second component using the automatic wavefront annotation algorithm. This algorithm considers the maximal negative slope of the unipolar signal within the window demarcated by the bipolar signal. Therefore, when on the annular plane, but distant from the AP location, the algorithm annotates the late ventricular signal. As the catheter is moved closer to the AP location, the ventricular signal appears earlier in time and, at the exact AP location, both the AP signal and the ventricular signal with the maximal precocity are seen. This is where the wavefront algorithm correctly annotates the maximal precocity corresponding to the AP location on the activation map (red focal area).
Overall, the use of EA mapping permitted to minimize the fluoroscopy time, which was zero in the right atrial procedures or when a PFO was present, and it was near zero in left atrial procedures with transaortic approach. This workflow has been tested with the CARTO 3 system using wavefront annotation and pattern matching. As previously shown, 7 the wavefront annotation algorithm minimizes the variations that could be caused by low-quality signals, resulting in highly accurate maps. To our knowledge, the CARTO 3 system is the only EA mapping system with this feature.
Another feature used is the Parallel Mapping module, which enables simultaneous acquisition of 2 maps with different settings, such as 2 different windows of interest. With this feature we were able to acquire voltage and activation maps at the same time. The voltage map enables quick identification of the valve plane and any potential alteration in the substrate, whereas with the activation map we successfully localized the target ablation zone. Again, to our knowledge parallel mapping is available only with the CARTO Prime software version v7.
As illustrated by the case studies, the new workflow has great potential in the diagnosis and treatment of overt AP, for many reasons. It increases the precision of EA mapping, reducing dramatically the area of AP localization on the reconstructed map. This allows focusing the ablation to a smaller zone of cardiac tissue, shortening the time of RF delivery, and making the ablation safer (possibly lowering risk of complications such as pericardial effusion or tamponade). Moreover, it can reduce the number of catheters used during the procedure. In selected cases only 1 mapping and ablation catheter may be sufficient to localize the AP and perform the ablation, thus diminishing the risk of vascular complication, especially for young patients.
To conclude, the use of EA mapping helps reduce fluoroscopy time, if not avoiding it (for right-sided APs or in the presence of PFO), being useful in select categories such young patients and women of childbearing age. Additionally, the present workflow allows for fast mapping time with automatic signal annotation, avoiding manual annotations, which are more prone to inaccuracies.
[fig] KEYWORDS: Accessory pathway; Activation map; Automatic annotation; Catheter ablation; SmartTouch (Heart Rhythm Case Reports 2021;7:674-678) [/fig]
[fig] Figure 1: Case 1. Top left: CARTO 3 voltage map of the right atrium in a left anterior oblique (LAO) projection; top right: CARTO 3 activation map showing the target point in red in LAO. Both maps have 190 points. On the bottom the accessory pathway block is visible after ,4 seconds of radiofrequency ablation. CS 5 coronary sinus; IVC 5 inferior vena cava; SVC 5 superior vena cava. [/fig]
[fig] Figure 2: Case 2. From left to right: the electrocardiogram (ECG) and electrogram (EGM) signals at the target site; posteroanterior (PA) view of the CARTO 3 activation map; ECG and EGM signals during radiofrequency ablation showing accessory pathway block after 1677 ms. [/fig]
[fig] Figure 3: Case 3. At left, CARTO 3 activation map (posterior view) with red zone indicating the accessory pathway (AP) location where the AV signals recorded by the ablation catheter (MAP 1-2) show the maximum fusion. On the right, the signals during radiofrequency ablation with AP block after ,2 seconds. [/fig]
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Development of a Codebook of Online Anti-Vaccination Rhetoric to Manage COVID-19 Vaccine Misinformation
Citation: Hughes, B.; Miller-Idriss, C.; Piltch-Loeb, R.; Goldberg, B.; White, K.; Criezis, M.; Savoia, E.
# Introduction
The COVID-19 pandemic has arisen in a period of increasing vaccine hesitancy in the United States. Vaccine hesitancy is defined as a delay in the acceptance of a vaccine or the outright refusal to take a vaccine despite its availabilityVaccine hesitancy is of particular concern when a vaccine is the primary method to mitigate the spread of a serious disease. Prior to the beginning of the COVID-19 pandemic, vaccine hesitancy was declared one of the top ten threats to global health by the World Health Organization. The pandemic emerged shortly after a 2019 measles outbreak, which has since been tied to parental reluctance to vaccinate schoolchildren. This was the worst outbreak of its kind since 1992and since a historic low of 0.15 measles cases per million in a 2002outbreak.
Several national opinion polls have found a significant portion of the US population is hesitant to take a COVID-19 vaccine. The prevalence of COVID-19 vaccine hesitancy has ranged from a quarter to half of the US population depending on the time in which surveys were conducted, reflecting the ongoing challenge of addressing vaccine hesitancy in the pursuit of herd immunity.
## The role of online fora and social media
Multiple mechanisms can contribute to the spread of vaccine hesitancy, among which online forums play an important role in persuasion. In the past, persuasion might have occurred via a "two-step flow," in which opinion leaders were integral to the adoption of people's worldviews. However, with the growth of increasingly personalized and automatically customized content channels offered on the web, some scholars have observed the emergence of a complementary "one-step flow" of persuasion. In this "one-step flow," web users are influenced directly from an online experience that has been algorithmically tailored to address their interests-and psychological vulnerabilities. The present study is informed by the hybrid theory of Hilbert et al., which argues that while persuasion has indeed become less interpersonal, and more automated and direct, online influencers and trusted offline sources still play a crucial role in shaping opinion.
Several authors have examined how social media platforms contributed to vaccine hesitancy prior to the COVID-19 outbreak. Amplification of vaccine hesitancy online has continued in light of development of the COVID-19 vaccine. Studies reviewed by Puri and colleaguesdescribe how anti-vaccine content frequently generates greater user engagement than its pro-vaccine counterparts on Facebook. Singh and colleaguesfound that low-quality sources of misinformation on COVID-19 were more commonly retweeted than those with high-quality information. In a comparative analysis of the spread of misinformation on five social media platforms (Twitter, Instagram, YouTube, Reddit, and Gab), Cinelli and colleaguesanalyzed more than 8 million comments and posts over a time span of 45 days, to model the spread of misinformation and demonstrated that social media platforms can serve as amplifiers of misinformation.
Vaccine hesitancy at large often arises in response to real-world events that create a window of opportunity for narratives countering vaccine uptake. According to Betsch et al, even limited and short-term exposure to anti-vaccine websites increased individual perceptions of vaccine risks. Buller and colleagueshave found that an individual's degree of engagement with Facebook posts on the HPV vaccine was predictive of greater vaccine hesitancy. Buller et al. hypothesize that on the social media platform, alleged risks associated with vaccines appear more immediate and tangible than risks associated with not receiving the vaccine (the success of vaccination is the absence of disease). Further, anti-vaccine messaging tends to be more focused on emotions and personal anecdotes with powerful imagery in contrast to the empirical strategies utilized by pro-vaccination literature and platforms. These emotional approaches tend to be more appealing to social media users and are consistent with other content that tends to be shared on social media.
## Types of anti-vax narratives and beliefs
Often vaccine hesitancy is treated by policymakers and risk communicators as a uniform belief; people are either hesitant to receive a vaccine or not hesitant. However, a subset of research has identified ways in which vaccine hesitancy is not a uniform belief system, but rather consists of several tropes and narratives that coalesce around common themes. In a review of 480 websites, all of which were promoting vaccine hesitancy, Moran and colleaguesfound several key values among those reviewed: choice, freedom, natural/holism, independence/individuality, and religion. These values were expressed in narratives related to topics ranging from lifestyle norms (references to alternative messaging or other health behaviors), mistrust in communicating authority, vaccine-related diseases, and vaccine ingredients counter to religion (pork products, for example). The most common value was "choice," appearing on one-third of the sample identified in the study.
Moran was the first to consider values or the "core" principles that may drive the more specific beliefs held by-or comments made by-individuals. The study by Moran and colleagues adds to an ongoing literature aiming to characterize the type of anti-vaccination content on the internet. These prior studies have identified a variety of specific comments or beliefs among content producers. Beanbuilds on the work of Davies and Wolfe and refers to specific values that emerge from this content: safety and effectiveness, civil liberties, and alternative treatments. More recent work by Johnson et al.has found that there is a greater number of smaller anti-vaccination groups online relative to pro-vaccination groups, allowing these groups to cover more "surface area" and attract the undecided to anti-vaccination messages. This "surface area" is further expanded when anti-vaccination groups create content focused on more than just vaccination-related issues, connecting to the other core beliefs these authors have described. Critically, Johnson et al. note that anti-vaccine messages tend to be framed as conversations between equals, that is, as peer-to-peer communication. Conversely, pro-vaccine content tends to take the form of expert-to-masses, top-down messages. Anti-vaccine communication, thus, bridges the gap between one-step and two-step flows of persuasion, perhaps the better to take advantage of online media's unique style of social ambiguity.
## Defining rhetoric and narrative in the context of ant-vaccine and covid-denial content
Whereas these prior studies analyzed the quality of information related to vaccine hesitancy and the values that motivate hesitancy, the study we conducted focuses on the narrative tropes and rhetorical styles of anti-vaccine content. Narratives combined with particular rhetorical strategies can be more or less influential. This study adopts a simple definition of the term "narrative" predicated on the features of change and temporality. Dahlstrom provides an excellent definition of narrative in the context of science communication to mass audiences as "a particular structure that describes the cause-and-effect relationships that take place over a particular time period that impact particular characters"(p. 13614). Our simple definition is in keeping with established scholarly uses of the term.
For the purposes of this study, a "trope" is understood to mean a subsidiary element of narrative, indicative of the larger narrative or narratives in which it is contained-narratives which might be vague, only partially articulated, or even based on mysterious/absent plot, characters, and/or meaning. Tropes, therefore, "are about relationships, and never about the term in itself"(p. 3). Tropes, in other words, definitionally refer to something beyond the context in which they appear. A "narrative trope," therefore, is some semiotic element-visual, aural, or written-which connotes a larger story or worldview.
For the purposes of this study, a "rhetorical strategy" refers to those modes of persuasion that can be separated from narrative appeals, which do not in-and-of-themselves indicate a larger story or narrativized worldview. These generally comport with the classical categories of rhetorical appeal-ethos (authority), pathos (emotion), and logos (logic)-and with the Platonic critique of rhetoric such as vulgar and manipulative. On one hand, we look to the classical definitions because contemporary definitions of rhetoric are diverse and unresolved. This study takes an expansive view of rhetoric, to include audio-visual as well as written and verbal rhetoric. This is in keeping with the common scholarly use of the terms and concept. Narrative tropes and rhetorical strategies, thus, combine to form persuasive messages, which may be either anti-social (as in this study) or prosocial (as in the public health messaging described below).
## The efficacy of narrative and rhetorical persuasion vs. appeals to reason
While the literature is not uniform in its conclusions, there is evidence to suggest that narrative and perspectival persuasion can be more effective than factualargumentative approaches. Research shows that audience absorption into a narrative reduces the audiences' capacity to form counterarguments against messages contained in the narrativeand tends to strengthen the persuasiveness of weak arguments and appeals. As per Maertens, Roozenbeek and Van der Linden, messages addressing a persuasion technique rather than a specific factual claim offer the potential "to achieve broad-spectrum resistance against manipulation," which factual rebuttal might not(n.p.).
Braddock's work in the field of attitudinal inoculation likewise suggests that attention to form yields greater negative attitudinal reactance against undesirable belief or behavior than attention to content. Ratcliff and Sun argue that narrative's effectiveness comes from its ability to circumvent psychological resistance, so that "when a persuasive message is embedded in the story and/or carried by the characters, persuasion occurs to the immersed, less critical, and less defensive"(p. 414). Additionally, literature from the field of violent extremism studies suggests that individuals are most often brought to false and conspiratorial perspectives not by rational decision making, but through a search for emotional and social fulfillment.
Work in the field of public health messaging has demonstrated that messaging that focuses on narrative and rhetoric (form) tends to yield better persuasive outcomes than messaging that focuses on facts alone (i.e., content). A variety of studies from the field of advertising, media, and communication studies suggest that narrative messaging yields more effective outcomes than analytic and informative messages. As a practical matter, Deng et al. found advertising messaging related to COVID-19 deployed "transformational" strategies (that is, appealing to emotions) over informational ones by a ratio of nearly two-to-one.
## Study objective
In anticipation of the unfolding COVID-19 vaccination campaign, this study sought to identify narrative tropes and rhetorical strategies in the emerging vaccine hesitancy discourse. To this end, we developed a codebook of the most commonly found antivaccination themes, both general and specific to COVID-19. We did so with the intent of providing public health communicators and social media moderation teams with a codebook of compelling anti-vaccination themes, against which counter-messages might be tailored. This work contributes to the nascent literature on COVID-19 vaccine hesitancy beliefs and the ways in which anti-vaccine messages are persuasive.
# Materials and methods
# Manual coding methodology
This study employs a simple manual coding approach to identify the narrative tropes and rhetorical styles described above. This method uses both inductive and deductive analytic approaches to gather its corpora. These corpora are analyzed both inductively and deductively, in two rounds of independent manual coding by a team of three subject area experts. Each team member's codes are compared so as to substantiate their relevance, and then consolidated "to construct more abstract concepts"(p. 2) in keeping with the action research approach of identifying salient narratives and rhetorics. This final body of codes is then assessed quantitatively to determine code frequency and distribution, that is, both how many times a code appeared and how many distinct pieces of content it appeared in. This methodological approach is a conventional and widely accepted tool, which excels at identifying "summative, salient, essence-capturing, and/or evocative attribute[s]"(p. 4) in mixed-media corpora. This subtlety and complexity is not so easily gleaned using more quantitatively centered methods and large datasets. However, similar to any method, it does possess shortcomings. These gaps, and the future avenues of study they suggest, are detailed in the "Future Directions" section at the end of this article.
## Data sources
Data collection aimed to develop corpora (i.e., large collections of codable content) for two separate rounds of coding. These corpora were developed via purposeful sampling methodology. Using hashtag and keyword searchers, a team of subject matter experts identified 20 channels (i.e., bounded sources of content, such as a social media account), which appeared to contain a high degree of anti-vaccine content and/or COVID denialism. For each round, five distinct channels were selected for preliminary coding. Only publicly available, English language media were selected, both to comply with platform terms of service and to focus coding efforts on content available to the uncommitted and vaccine hesitant. Geography was not considered, due to the transnational availability of the channels that were surveyed. Channels were selected according to the criteria of (1) offering 50-100 pieces of codable content from the past six months, (2) covering written, video, and/or image/meme media, (3) related to COVID skepticism/denial, anti-vaccine ideology, and/or conspiracism, and (4) discursive significance, based on quantitative and qualitative evaluation.
The five corpora coded in this first round included: the Plandemic propaganda documentary, the Vaxxed propaganda documentary, official posts from the Facebook group of the anti-vaccine and COVID-skeptic organization The Children's Health Defense (going back three months, excluding comments), the "fake news" blog OffGuardian's "COVID Factchecker" vertical (comprising seven blog posts), and the Instagram account "COVID Funny Memes" (constituting 100 pieces of relevant content, excluding comments). The discursive significance of these channels was established based on the following criteria: the films Vaxxed and Plandemic enjoyed widespread distribution on online platforms; the Children's Health Defense organization's Facebook page has over 145,000 followers and is headed by celebrity vaccine detractor Robert F. Kennedy Jr.; the "Funny COVID Memes" page had only approximately 5000 followers, but was selected for its apparent ideological neutrality (i.e., both vaccine-denialist and pro-vaccine content appeared in roughly equal proportion); the OffGuardian blog represented the least widely consumed inclusion, ranking 74,278 in global internet engagement according to Alexa rankings-it was chosen in order to include a text-only coding source and due to its clear COVID/vaccineskeptic position.
Sampling for the second round was likewise purposive, with a focus on capturing media and audiences that might have been missed during Round 1. Channels that spoke to minority demographics were specifically sought out. Once again, five channels were selected for coding based on the same criteria as Round 1. The five corpora constituting the second round of coding were: the Twitter account of Joyce Brooks (a representative of the Denver NAACP and vaccine denialist), the Twitter account of Toby Rogers (a popular and prolific anti-vaccine figure with a following of 29.7k at the time of sampling), the YouTube video "A Message to Aussie Muslims" by Sufyaan Khalifa (urging resistance to COVID public health measures), the Gardasil Girls "vaccine injury" Instagram account (including top comments), and the Vaccines Uncovered Instagram account. For all four non-video accounts, 50 individual posts, including retweets and reposts, were collected.
The purpose of this selection methodology was not to select the most popular or influential COVID/vaccine-skeptic/denialist channels, but to select a spread of channels that could reasonably be expected to move the research team toward theoretical saturation. Saturation was defined by the research team as that point at which "additional data do not lead to any new emergent themes"(p. 135).
# Coding methodology
Round 1 corpora were loaded into NVivo, and each member of the four-person codebook team independently coded each corpora. Codes were then compared and consolidated. Infrequent codes were set aside. In the end, preliminary coding produced 34 unique codes pertaining to the narrative tropes and rhetorical strategies that circulate in the anti-vaccine and COVID-skeptic media.
These initial 34 codes were then applied to the second round of corpora. Round 2 corpora were coded according to the preliminary codebook produced in Round 1, with new codes added as deemed appropriate by the coding team. The coding schema developed by each individual member of the codebook team were compared and consolidated. An additional 29 codes were identified during the second round of coding. However, these codes were either found only sporadically or could be consolidated into existing codes. These codes were then analyzed according to an action research methodology approach.
The rates of code appearance across corpora were analyzed in comparison with their frequency within corpora. Based on repetition of themes, it was determined that codes pertaining to general audiences (i.e., not demographically specific) had reached a point of adequate saturation. However, in keeping with action research methodology, some relatively infrequent codes were kept. This was done in order to address codes specific to minority demographics.
The codebook team drafted detailed explanations of the codes and then grouped them according to whether they referred to a narrative trope or a rhetorical strategy, based on the criteria outlined in the literature review. Narrative tropes were further organized according to the implicit antagonist of the narrative. Implicit antagonists were determined based on close reading of content from which these codes were derived. Rhetorical strategies that frequently occurred alongside specific narrative tropes were noted. Examples from the corpora were assigned to each code in order to further illustrate.
# Results
Of the final codebook's list of narrative tropes, five patterns represented more than half of the coded items: "Corrupt Elites," "Vaccine Injury," "Sinister Origins," "Freedom Under Siege," and "Health Freedom." Of the final codebook's list of rhetorical styles, four patterns represented just over 40% of all coded rhetorical items: "Think of the Children!" "Do Your Own Research," "Heroes and Freedom Fighters," and "Panic Button" (see. All nine of these codes appeared across all four major platforms from which data were sampled: YouTube, Twitter, Facebook, and Instagram. However, it should be noted that salience cannot be determined solely by the frequency with which a code appeared either within or across channels. In keeping with this study's action research methodology, other factors, such as interaction with other narratives and rhetorics and subjective judgements of affective intensity, were taken into account when determining salience. In total, the results of our coding show that online, English-language, anti-vaccine, and COVID-denial content can be classified according to twenty-two narrative tropes and sixteen rhetorical strategies (see. The twenty-two narrative tropes can further be classified as directed toward five major types of antagonists: (i) the government, (ii) the medical establishment and political/economic elites; (iii) mainstream (and implicitly, pro-vaccine) society at large; (iv) an entirely unspecified "shadowy villain"; (v) the vaccine itself. Some additional unclassified antagonists form a miscellaneous category.
## Corrupt elites
Populist framing of a righteous majority versus corrupt elite. Elite perceived as forcing lockdowns and health practices for their own financial gain (e.g., big pharma) and/or power.
## 20.4%
## Vaccine injury
A catch-all for all of the harms the vaccine can do to you, from physical deformities to mental illness to microchips that violate your autonomy/privacy.
## 12.1%
Sinister Origins
The people who intentionally created the COVID vaccine are shadowy and suspicious. Geopolitical powers and intelligence agencies are likely implicated.
## 10.4%
Freedom under Siege
Common rights such as speech, assembly, or autonomy are being stripped from you! This claim attempts to hijack feelings of protection, vulnerability, and the sacred.
## 9.3%
Health Freedom
Frames public health as a matter of individual freedom rather than collective responsibility. "My body, my choice" misapplied to vaccines.
Tied to religious freedom/freedom of speech.
7.8%. Cont.
## Rhetorical strategies explanation prevalence
Think of the Children! Frames this as an advocacy campaign for protecting children. Uses emotionally affecting (manipulative) images of cute children..1%
## Do your own research
States a conclusion and then urges the reader to research the reasons why. Visual clues lead to building arguments in favor of a predetermined anti-vax position.
## 8.4%
Speaking Truth to Power Doctors, nurses, and other professional "experts" speaking against COVID alarmism are brave whistleblowers, courageously bringing truth to the people.
## 8.1%
Panic Button Audio and visual cues intended to spark alarm, fear, or disgust, such as ominous music and images of needles or malformations. 7.2%. Rhetorical strategies.
## Absurd!
Rhetoric that holds up public health practices and cultural expressions of care/anxiety over COVID-19 to ridicule. This includes ridiculing both experts and laypeople, sometimes through misrepresentation (see "Mountains and Molehills"). Key to this rhetorical strategy is an overall tone of mockery and/or contempt.
## Appropriating feminism and/or womanhood
Anti-vaccination messages that appropriate the language and values of feminism, such as claiming that vaccine resistance is the positive moral equivalent of advocating for reproductive rights; also, sometimes appropriating themes of femininity, the stereotypes of maternal wisdom and nurturance.
## Brave truthteller
This strategy frames the speaker as brave for publicly voicing their anti-vaccine opinions, despite the potential for public backlash, parenting judgment, or criticism from supposed experts. This strategy celebrates vaccine resistance by depicting its messengers as heroic in their stand against the establishment, akin to a whistleblower standing up to corruption. Sometimes, this takes the form of the speaker themselves claiming he or she is brave; other times, someone else's bravery is highlighted. In vaccine-hesitant and -resistant spaces, the bravery often pertains to standing up to others' ridicule, or to the implication that one is a bad parent.
## Dyor (do your own research)
This approach often states a conclusion contrary to mainstream beliefs or scientific consensus, and then urges the audience to research the reasons why the conclusion is correct. This leads inquiring minds to build their own arguments in favor of a predetermined position. Other times, this strategy is deployed to avoid answering questions or engaging in debate. The implication is that if audiences reach the correct conclusions (i.e., the ones the speaker asserted), then they have done good research. If they disagree, their research must have been bad.
## Epic significance
The struggle against vaccination is framed as one of global, historical, or even mythic proportions. Hyperbolic rhetoric and superlatives are used to convey that this threat is profound enough to change the world, to enshrine the power of a corrupt elite-or to imperil the most vulnerable among us (children). In addition to the exaggeration of the threat posed by vaccines, this strategy positions the audience as capable of, or even obligated to, participate in this epic quest for justice.
## A global movement/sleeping giants
Rather than inflating the threat of the vaccine to epic proportions, this strategy inflates the anti-vaccine movement itself. The voices of ordinary people all around the world are depicted as speaking as one, a unified, grassroots groundswell against evil. Sometimes anti-vaccine and anti-public health movements are framed as just the beginning of a groundswell that addresses other conspiracies. This strategy employs a populist frame that all over the world, good ordinary people ("just plain folks") are ready to rise up and take back the power over their own lives.
## Health freedom
This strategy frames public health as a matter of individual freedom rather than collective responsibility. Sometimes, this even borrows from the language of women's reproductive rights, re-appropriating concepts such as "my body, my choice" to vaccines. This is sometimes related to religious freedom (vaccines) or freedom of speech (anti-mask).
## Hijacking familiar formats
In general, memes work by using a familiar format to make a new joke or prove a new point. Rhetorical strategies that hijack formats adopt popular formats that are associated with harmless humor and use that familiarity to lower our emotional and cognitive defenses. With its audiences' critical defenses down, it then presents its manipulative message. Beyond memes, this also applies to TV, film, and game formats that are appropriated for propaganda. This strategy also includes "hashtag hijacking," where vaccine misinformation will be tagged with hashtags relating to less controversial issues and groups, such as #blacklivesmatter or #americancancersociety. Hashtag hijacking is particularly effective for exposing newcomers to anti-vaccine messages.
## Intersecting social justice
Rhetoric similar to this frames vaccine skepticism alongside social justice issues or frames vaccine skepticism itself as a social justice issue in its own right. Sometimes, it may invoke systemic racism or the abuse of minorities by the police to imply that the medical establishment is similarly racist. It may frame its argument more generally, too, presenting vaccine resistance as a matter of either civil rights or religious conscience. It may describe non-vaccinators as "health minorities" or "dissidents."
## Lovebombing
Allies and fellow "truth-tellers" are showered with affirmation, accolades, validation, and compliments. This often includes emotive videos or images celebrating allies for their cause as heroes or persecuted saints (see also: "Sleeping Giants").
## Mountains and molehills
Risks and benefits to vaccines are presented with intentionally distorted proportions. Extremely small risks (e.g., negative reactions to a vaccine) are framed as catastrophic and universal. Simple and far-reaching solutions (e.g., mask mandates) are presented as onerous and ineffective. This strategy distorts the risk/reward calculus of vaccines in order to seed doubt, often by emphasizing fringe or outlier cases of vaccine injury.
## Panic button
A common rhetorical technique that uses audio and visual cues intended to spark alarm, disgust, confusion, squeamishness, anxiety, or dread in audiences. Ominous music can be used to indicate that viewers should be worried or mistrustful about what is shown to them. Images of hypodermic needles, malformations purportedly caused by vaccines, or forced vaccinations are depicted in ways that evoke fear and/or disgust.
## People are saying
This strategy states or implies that "many" people feel a certain way, evoking a social norm against vaccination. The strategy depicts those who agree with the speaker as good people, and those who disagree as fearful conformists. It may imply that evidence exists simply because other people are allegedly saying it, even though there is no actual evidence presented. Otherwise, it may rely on testimonies, first-hand accounts that usually emphasize emotion over facts, and may or may not actually be true.
## Question begging
A technique that poses questions designed to set up a narrative, as opposed to asking questions for objective journalistic purposes. This strategy asks a series of questions that lead to a specific anti-vaccine answer, while framing the conversation as objective and inquisitive.
## Think of the children!
This rhetoric suggests that anti-vaccine advocacy is not about what activists want for themselves, but rather what is best for children. Arguments are framed so as to position children's exaggerated physical vulnerability and moral purity as the decisive factor in assessing risk. It often uses emotionally affecting (manipulative) images of cute children.
## Trappings of authority
Often a visual rhetoric, this strategy uses symbols of authority and expertise to give added weight to an argument. A speaker might be in an office full of books. They might be in a doctor's office. They might be expensively dressed. The interviewer or director might refer to them with exaggerated deference. Sometimes, their credentials are presented as if they were very impressive but, when examined more closely, are spurious or over-inflated.
## Narrative tropes
Of the twenty-two narrative tropes coded, four key common narrative tropes were: "Vaccine Injury," "Corrupt Elite," "Heroes and Freedom Fighters," and "Sinister Motives." Each of these narrative tropes addressed a separate antagonist, representing all categories of antagonist-the Government/the Medical Establish/Media, Society at Large, a Shadowy or Unknown Villain, and the Vaccine Itself-except for "Misc./No Clear Antagonist."
The narrative of "Corrupt Elite" was the most frequently coded narrative. This offered a standard populist appeal in which an innocent put disempowered "silent majority" suffer under the tyranny of a powerful and corrupt minority. In a process of circular logic, the fact that the government, medical establishment, and high-profile cultural figures support such measures as lockdowns, masks, and vaccines is taken as sufficient evidence that these measures are untrustworthy. The narrative of a corrupt elite was very frequently seen with related but distinct codes. "Follow the Money," for example, often co-occurred, offering financial motives for elite corruption. "Sinister Motives" offers malevolent and even supernatural explanations for elite missteps. The "Unaccountable Elites" narrative frames government and the medical establishment as immune to consequences from the bad or incompetent actions; it is motive agnostic, but argues that whatever their motives, elites bear no consequences for the mistakes and, therefore, are always to be mistrusted and disobeyed. These, along with other compatible narratives, point to a larger metanarrative that pits potential vaccine recipients against all of the social institutions working to provide them with a vaccine.. Narrative tropes (organized by primary antagonist).
## Antagonist: government/establishment and elites:
Narratives 1-8 are framed in such a way as to villainize experts, authorities, and figures of cultural influence. These "elites" consist of groups such as the medical establishment, governments, media, and press.
## 1984
This narrative depicts the COVID pandemic and all public health measures associated with it as the final few steps toward a maximally repressive global government. It presents a "domino theory," in which free speech, freedom of religion, and freedom of travel will soon be abolished. Every time a new public health directive has been passed, it says, many vaguer, but far worse, oppressions are sure to come next.
## Alarmist authorities
This narrative presents a distorted pattern of events in which authorities' warnings and measures against COVID are overblown. (see Fluffing the Curve and Follow the Money)
## Censored!
Digital platforms and social media are portrayed as actively engaged in "censoring" advocates of "health freedom." This is often framed as a David vs. Goliath scenario where powerful companies conspire against brave individuals speaking truth to power. This is described in the language of a grave injustice.
## Corrupt elite
This narrative is a standard populist appeal. The world can be divided into a corrupt elite and a righteous majority. The corrupt elite is on the side of lockdowns and mandatory masks/vaccines. The fact that the elite favors these lockdowns, masks, and vaccines is taken as more than sufficient evidence that they should not be trusted. So the reasoning goes: the elites must be corrupt, because they are pushing an untrustworthy and potentially dangerous medicine.
## Fluffing the curve
This narrative argues that officials are misrepresenting the numbers of COVID injuries and deaths, or that doctors are somehow incentivized to report more deaths. Perhaps they are doing so to ensure profits (see "Follow the Money"), or perhaps to instill fear and control (see "1984," "Sinister Motives"). This category also includes "apples to oranges" comparisons of patient categories, different diseases' mortality rates, vaccinated vs. unvaccinated health outcomes, and more.
## Follow the money
This narrative paints the COVID pandemic as an unprecedented opportunity for corporate looting and medical profiteering. Additionally, anything that points to more robust public health initiatives is almost certainly a set-up for crony handouts and panic-driven marketing. There is big money in medicine, this narrative says, and for media giants, there is big money in making people "panic-watch" and "doomscroll." These are stories in which powerful men will do whatever it takes to compete and aggrandize their wallets and ego-whether it means lying, neglect, withholding care or resources, or plain out killing. There is a specific sub-category that describes claims made against Anthony Fauci regarding supposed fraud. Most famous is the "HIV Scandal" involving a series of vague accusations of silencing patients, academics, and scientists to uphold a Ponzi scheme related to HIV treatment protocols.
## Freedom under siege
This narrative paints a story in which common rights such as speech, assembly, or possession of some entitled object are being stripped from citizens. This claim attempts to hijack feelings of protection, vulnerability, and the sacred. Can also be framed with the key words, "Religious and Philosophical Exemption."
## Unaccountable elites
These narratives are framed around the assumption that doctors, politicians, and the media will never have to account for their lying or incompetence. So the story goes: if they have no skin in the game, then why should we believe a word they say?. Cont.
Antagonist: Society at Large: Narratives 9-12 pit anti-vaccine advocates and COVID denialists against society in general or specific elements of it, such as our public political discourse or areas where racial disparities are acutely felt.
## Heroes and freedom fighters
Here, doctors (and "doctors") speaking out against vaccine injury or COVID alarmism are brave whistleblowers, acting at tremendous personal and professional risk to bring the truth to the people. The people protesting public health measures are painted as the moral and ideological equivalent of Soviet dissidents, the founding fathers, and the Arab Spring all rolled into one. This narrativizes the "Brave Truthtellers" rhetorical strategy by imbuing it with specific protagonists and struggles.
## Erasing poc
This narrative argues that people of color are shut out of public debate over vaccination, that their voices are dismissed, or they are tokenized and only deployed when it is convenient for the white and powerful. It might also argue that people of colors' rights to "health freedom" or their experiences of "vaccine injury" are invisible due to systemic racism in the medical system. It usually accompanies tropes such as "Racist Medicine" or "POC Injury." It is an example of how effective anti-vaccine narratives can be essentially correct, but still point toward false and damaging conclusions.
## Racist medicine
This narrative points to the real history of medical abuse of minorities in the US and elsewhere and implies that minorities should, therefore, mistrust what they hear about COVID and vaccines. Usually, no specific threat or conspiracy is articulated. The history is described and the connection with the present day is left implicit, but clear (see also: Intersection with Social Justice, Erasing POC, POC Injury).
## "you made it political!"
This frames the conflict between vaccination and non-vaccination as a partisan political issue. On one hand, it might state that pro-public health voices are the ones making this political, when it is actually a matter of common sense, religious freedom, or personal choice. On the other hand, this narrative category might take an explicitly partisan tone, for example arguing that former President Donald Trump was heroically battling big pharma and a corrupt elite.
## Antagonist: shadowy villain:
Narratives 13-14 do not offer a specific villain, but implicate an extremely powerful and mysterious agent whose means and motives are unknown-perhaps beyond comprehension. Conspiracy theories that verge on the supernatural often framed their antagonist in these terms. These demonstrate that narratives can be based around an absence or unarticulated mystery (see literature review).
## Chinese virus
These stories claim with absolute certainty while lacking in substantive proof that the virus was created or leaked from the Wuhan lab in China. These tropes are distinct from legitimate inquiry into a possible "lab-leak hypothesis," because of the narratives that they indicate. Sometimes, those narratives claim that a virus cannot mutate that quickly, or that COVID is a powerful bioweapon and the idea that we can easily stop it with masks or a vaccine is laughably naïve. These narratives are highly compatible with long-existing anti-Asian stereotypes as a sinister "enemy within" Western countries.
## Sinister motives
The people behind the COVID vaccine are described as shadowy and suspicious. Geopolitical powers, pharmaceutical corporations, and intelligence agencies are likely implicated.
## Antagonist: the vaccine itself:
Narratives 15-19 focus on the harm they imagine a COVID vaccine will inflict. Unless tied to another narrative or rhetoric specifying additional antagonists or personifying the vaccine, these narratives offer an antagonist that is impersonal and without motive.
## The perfect family
These narratives are often framed around anecdotes of supposed vaccine injury. Children are presented as perfect angels, baby geniuses, junior Olympians, etc. Parents are presented as bursting with pride, ready for a smooth, normal, American (or English or Australian or w/e) life. Then came the vaccine, and its injury. Then came the never-ending tribulations. The dream is long dead.
## Poc injury
This narrative states that ethnic minorities have congenital conditions which allopathic medicine does not properly consider during the development of treatments and vaccines. One example is the claim that African Americans, particularly boys, have stronger immune systems that are more reactive to vaccines. While the coding team did not encounter similar messages targeting women of any race, it seems possible that women's higher rates of autoimmune disorder, and historic mistreatment in medicine, could underpin similarly pseudoscientific theories (see also "Racist Medicine").
## Rushed vaccine
These narratives say that the COVID vaccine has been rushed to market without proper testing, that it could not have gone through trustworthy safety protocols, and that the public cannot trust that it will be safe.
## Unknowable dangers
These narratives assume that we should apply the precautionary principle to dangers associated with preventing COVID (i.e., vaccines) but not to COVID itself (e.g., the danger is overblown, go to the pub!) (see also: Mountains and Molehills). This is distinct from the Vaccine Injury narrative, as it focuses on vague potential future outcomes, whereas Vaccine Injury focuses on specific, and often present-day, claims of injury.
## Vaccine injury
A catch-all term for all the bad things vaccines can do to you, with no legitimate causal link required. Extremely common.
## Miscellaneous/no clear antagonist:
Narratives 20-22 either did not present a clear antagonist or were not consistent enough in their imagined antagonists to effectively classify.
## All-or-nothing
These narratives cast their heroes and villains as either all trustworthy, good, and "on the right side" or else dangerously misguided, stupid, or evil.
## Imminent threat
Narratives of this sort warn their audience that "time is running out," and something terrible is either happening or about to happen very soon. This threat could be specific (e.g., a law being debated that would mandate vaccines for public school attendance), or it could be vague (e.g., the end of America). The warning is very frequently accompanied with some call to action, such as calling your congressman or evangelizing in favor of anti-vax messages.
## Overblown risk
These narratives dismiss risks associated with COVID as overblown. They sometimes misuse statistics to reach this conclusion, such as comparing high-risk populations' flu mortality rates to low-risk populations' COVID mortality. Most often, these narratives center around an emotionally dismissive claim of others' alarmism. This is distinct from the Alarmist Authorities code, as it addresses a more general cultural alarmism that may originate in not-elite sources.
"Vaccine Injury" appeared to be the most salient of narratives, though not the most frequent. The code was a catch-all concept describing all negative outcomes (almost exclusively imagined or untrue) associated with taking a vaccine. Examples of this narrative rarely provided a clear causal link between vaccination and injury. Instead, it most frequently juxtaposed a claim to having been vaccinated alongside a claim to injury. These injuries were often vaguely described and only infrequently accompanied by claims to an actual diagnosis of malady. Messages conveying this narrative frequently used the "Panic Button" style of audio-visual rhetoric, in which images of needles, crying infants, unsettling sounds, or unattractive colors are employed to produce feelings of disgust and unease.
A third popular narrative, "Heroes and Freedom Fighters," is compatible with populist framings, presenting anti-vaccine or COVID-denialist medical doctors (as well as chiropractors and naturopaths) as brave whistleblowers, risking their reputations and careers by speaking truth to power. This narrative often coincided with rhetorics of "Brave Truthteller," "Sleeping Giants," and "Health Freedom," presenting the people organizing against public health measures as the moral equivalent of pro-democracy dissidents.
## Rhetorical strategies
Of the sixteen rhetorical strategies coded, four key, common rhetorical strategies were: (i) the "Brave Truthteller," (ii) "Do Your Own Research (DYOR)," (iii) "Mountains and Molehills," and (iv) "A Global Movement/Sleeping Giants". Each of these rhetorical strategies was present across multiple categories of narrative antagonist. For example: the rhetorical strategy labeled "Brave Truthteller"-in which a speaker claims to be speaking a dangerous truth that "the establishment" or society at large is suppressing-was significantly present in all four categories of antagonists and even in those relatively few narratives with no clear antagonist.
Another common rhetorical strategy that is directed toward multiple protagonists is "DYOR" or "Do Your Own Research." DYOR works by trying to empower the audience to develop their own bodies of evidence and methods of reasoning in order to reach a preordained conclusion. Sometimes, this leads inquiring minds to build their own arguments in favor of a predetermined position. Other times, DYOR offers vaccine denialists and skeptics a means to avoid answering questions or engaging in debate: they may simply demand that the interlocutor should do their own research. If the interlocutor reaches the correct conclusions (i.e., anti-vax conclusions), then they have done good research. If they still disagree, so the reasoning goes, their research must have been bad.
A third popular category is what we refer to as "Mountains and Molehills." In this rhetorical strategy, vaccines' risks and benefits are presented without a proper sense of proportion. Extremely small risks (e.g., negative reactions to vaccines) are framed as catastrophic and universal. Simple and far-reaching solutions (e.g., mask mandates) are presented as onerous and ineffective. The risk/reward calculus is thereby severely distorted. This strategy is used to seed doubt via emphasizing fringe or outlier cases.
Presenting COVID-19 vaccine resistance as a part of a global movement of "sleeping giants"-honest, everyday citizens who are on the cusp of rising up against an oppressive "global elite"-was a common rhetorical strategy that cut across narratives targeting all categories of antagonists. This rhetoric frames its accompanying narrative to suggest that the voices of ordinary people all around the world are articulating that narrative as a unified mass. Anti-vax and anti-public health movements are presented to be just the beginning of this groundswell. All over the world, so the trope goes, self-conceived "ordinary people" are ready to rise up and take back the power over their own lives. This rhetoric taps into the populist persuasive strategies and schemata outlined above, which frame the pure, ordinary people against the nefarious, evil elite. In some cases, populist rhetoric intersects with nationalist rhetoric, arguing that only a stronger state can save ordinary people from bad elites. In other cases, these populist frames intersect with anti-government resistance, framing elites as not only out of touch with the needs of the pure, ordinary people but actively working against them in tyrannical ways that warrant uprisings, revolution, armed resistance, or even, a new civil war.
# Discussion
The scope of this study is intended to create a codebook of online English-language anti-vaccination narratives and rhetoric, so as to support government officials and civil society groups engaged in managing disinformation during the COVID-19 vaccination campaign. Further research into languages besides English tailored to address the regional cultural contexts that shade narratives and rhetoric are underway by members of this team, but beyond the scope of the present article. Local, national, and international governments, as well as civil society organizations, need to be prepared to manage the infodemic by promoting the timely dissemination of accurate information based on science and evidence, in particular to high-risk groups. This dissemination of accurate information should be both positive and defensive. That is, messaging campaigns must communicate both the latest in scientific understanding of COVID, its spread and prevention, and effective countermessages against misinformation. The codes identified by this study offer governments and civil society groups a catalogue of anti-vaccine message styles that may assist in the latter efforts.
Pro-vaccine audiences, the vaccine hesitant, and the wholly agnostic can generally be addressed as a single group. Anti-vaccine belief holders, a much smaller subset of the overall vaccine hesitancy spectrum, however, must have counter-messaging tailored specifically for them. This comports with the distinction between "prophylactic" and "therapeutic" counter-messaging. Evidence demonstrates that prophylactic exposure to counter-messaging would prompt hesitant, agnostic, and pro-vaccine audiences to develop their own counterarguments against misinformation and disinformation. Furthermore, these audiences would not need to be warned against every type of misinformation or disinformation they might encounter. Exposure to counter-messaging against one dimension of a mis/disinformation campaign confers resistance to other dimensions associated with that topic of mis/disinformation. This phenomenon is sometimes called the "blanket of protection". So, for example, an effective counter-message addressing exaggerated claims of vaccine injury can in theory be expected to confer some resistance to any other narrative trope or rhetorical strategy listed in this codebook.
The joint effects of targeted counter-messaging and blanket of immunity might best be mobilized through counter-messaging that addresses tropes and/or strategies that appear most frequently, and that are thematically linked to other denialist narratives and rhetoric. For example, the versatility of rhetorical strategies such as "Brave Truthtellers" may be both a cause and a symptom of their effectiveness; its versatility lends itself to a variety of narratives, while its repetition across narratives enhances its effectiveness through repetition. Counter-messaging that alerts audiences to the manipulative persuasion of the "Brave Truthteller" strategy will, therefore, be effective against a variety of misinformation and disinformation utilizing the strategy, as well as likely conferring further future resistance against the rhetoric and narratives that appear alongside it.
Similarly, "Do Your Own Research" is also a popular rhetorical strategy throughout conspiracy cultures, such as QAnon. QAnon disinformation networks have been shown to amplify anti-vaccination rhetoric and messaging, which raises important questions about the impact of overlapping persuasive rhetorical strategies in addition to amplification through hashtags or coordinated campaigns. By counter-messaging against transnetwork themes, a blanket of immunity may protect audiences from anti-vaccine and COVIDdenialist themes that are not described in this codebook or have yet to emerge.
Counter-messaging to address audiences who already hold undesirable viewpoints (i.e., therapeutic counter-messaging) is a newer and less well-established process than its prophylactic counterpart. A potential for backlash is always present in countermessaging campaigns. Additionally, while the so-called "backlash effect" (that is, the theory that factual counterargument entrenches false beliefs) has been credibly challenged, there is ample evidence that carelessly repeating false information can help spread it. Therefore, public health messaging that addresses anti-vaccination audiences, already hardened in their beliefs, must be preceded by especially rigorous testing. This codebook is presently informing a series of tests to determine the efficacy of public messaging that addresses some of the narratives and rhetoric found in it. The results of that study are expected to be available in pre-print by early summer 2021.
## Limitations and future directions
The present study has shed light on narrative and rhetorical patterns in anti-vaccine and COVID-denialist online media. However, the methods that made this study possible have also limited it in key ways. While the combined deductive/inductive coding process appeared to produce a saturation of codes, this cannot be definitively stated absent a corroborating study based on a large data sample. Such a study might be undertaken, using the present study's codebook as a deductive point of departure for a big data text mining and topic modeling project. It is possible that such an expanded study might reveal unexplored dimensions of anti-vaccine and COVID-denialist positions.
By the same token, this study was conducted with the intent of helping to inform public health messaging. A follow-up study is currently underway, which utilizes several of the codes mentioned in this article to create public health counter-messages. Among other questions, this follow-up study tests the efficacy of emotionally based countermessages relative to factually based counter-messages. It asks: is form indeed more salient than content to debunk health misinformation and disinformation? Here, more research is needed. Further work might also consolidate codes to supplement the "antagonist" categories offered here. Such a consolidation may help others to adopt these codes in independent public health messaging endeavors.
Finally, it is worth delving into the question of "why" these narratives and rhetorics hold such appeal. One might easily imagine a study of people holding anti-vaccine and COVID-denialist attitudes, which tests the salience of the narratives and rhetorics presented here. Such a survey could also address overlapping attitudes and worldviews to better ascertain the narratives indicated to believers by these tropes, and why such narratives and rhetorics are appealing.
# Conclusions
This paper reports on the qualitative classification of online, English-language antivaccination rhetoric about a COVID-19 vaccine. Our analysis of sixteen persuasive rhetorical strategies and twenty-two anti-vaccination messages directed toward specific antagonists is necessarily descriptive at this stage. We hope this work inspires additional empirical research to help illuminate the following questions: which of these rhetorical strategies and messages are most encountered online and for which demographic groups? Which of the messages carries the most persuasive appeal? How can persuasive rhetorical strategies be effectively countered in online spaces? How might different narratives and rhetorics appeal to audiences in different countries, different demographics and subcultures, and in different languages? Given the rise in populist movements across many countries, it seems possible that similar antagonisms would be articulated in anti-vaccine and COVIDdenialist media content. However, given the highly contextual and referential qualities of memes and other social media content, these antagonisms could reasonably be expected to assume significantly different expressive forms. To avoid the risk of blowback, it is essential that similar coding studies and counter-message testing be undertaken prior to the launch of public health campaigns addressing anti-vaccine and COVID-denialist misand disinformation.
# Data availability statement:
The data presented in this study are available on request from the corresponding author. The data are not publicly available due to concern for the privacy of the subjects whose public social media posts were used in the codebooking process. |
Efficiency of Human Resources in Public Hospitals: An Example from the Czech Republic
# Introduction
From an economic perspective, human resources in public health represent a highly specialized factor of production, irreplaceable in the short term. Public health and healthcare is an economically demanding sector where two thirds of financial resources in the individual organizations as well as the healthcare system are expended on labor force, and it is therefore essential to address the issues of the efficiency of human resources in order to ensure the sustainability and development of healthcare. The efficiency of human resources, especially health professionals, is a very topical, albeit controversial issue. Frequently mentioned, in this regard, is the disaccord between the efficiency, quality, and equality, but also the failure of the management of healthcare organizations and public administration, very often within the context of X-inefficiency [bib_ref] Human resource management and performance in healthcare organisations, Harris [/bib_ref] [bib_ref] A robust analysis of hospital efficiency and factors affecting variability, Leleu [/bib_ref]. Kozuń-Cieślak [bib_ref] The effectiveness of public investment in human capital, Kozuń-Cieślak [/bib_ref] state that the generally acknowledged positive relation between the human capital potential and prosperity cannot be verified because the investment into human capital in public health does not positively correlate with technical efficiency.
Human resources in public health are the executors of healthcare and the carriers of new and innovative therapeutic procedures, affecting the medical condition and quality of health of the population. Huanhuan et al. [bib_ref] A New Perspective for Improving the Human Resource Development of Primary Medical..., Huanhuan [/bib_ref] state that enough individual categories of workers and the process of lifelong learning of health workers is reflected in the improving quality of health services. According to the OECD (Organisation for Economic
## -
The first research question is based on the assumption that the least efficient human resource of a hospital are other workers, followed by the general nurses, while inefficiency is affected the least by the physicians.
RQ2: Is it true that big hospitals are more efficient than small and medium-sized hospitals?
- It is generally presumed that larger organizations are able to attain so-called returns to scale. However, consideration might also be given to the manifestations of the X-inefficiency phenomenon, linked with the monopolistic behavior of market operators and public administration and service organizations. The general basis of the monopoly position of the public service providers (hospitals) is their original, non-transferrable legal authorization for the provision of services and management of public funds and assets. It is therefore possible that, in practice, hospital consortia and big university hospitals manifest themselves as monopolies or oligopolies.
The article has five parts. The first part is this introduction; part two focuses on synthesizing the knowledge of the evaluation of the hospitals' technical efficiency and other healthcare according to the Data Envelopment Analysis Model (hereinafter the DEA Model); the third part deals with the research methodology, including the objective and research questions; part four presents the results of the calculations and analyses; and the fifth part contains the conclusion and discussion about the problem in relation to the results attained.
# Materials and methods
## Literature review: evaluation of technical efficiency according to the dea model
The healthcare service performance evaluation has become a fundamental basis for decision-making on the level of healthcare facility management as well as other healthcare policy actors, relating to strategic issues within a hospital's operation. The evaluation of health services has been addressed by a number of authors worldwide, a fact documented by a multitude of articles published on this subject. Multi-criteria methods are widely used for the evaluation as tools that are able to assess the efficiency of production units and show the opportunities for the improvement of inefficient units, but also to identify exemplary units. One of the tools able to determine the rate of technical efficiency of production units is the Data Envelopment Analysis Model. As shown in the articles specified below, an evaluation of technical efficiency over a multiannual period points to a greater stability of hospitals and thus to more relevant analysis results.
Hospital human resources are always modelled as inputs, while physicians and nurses are most frequently mentioned by the authors. But in some cases, they include all hospital employees among the human resources. Quite frequently, they combine human resources with other technical (e.g., number of beds, instrumentation) or financial (organizational) inputs in absolute or relative terms.
Pirani et al. [bib_ref] Hospital Efficiency Measurement Before and After Health Sector Evolution Plan in Southwest..., Pirani [/bib_ref] focused on the evaluation of the efficiency of public hospitals between 2012 and 2016. Their pivotal method was the output-oriented DEA Model positing variable returns to scale, while the input variables comprised the number of hospital admissions, the number of nurses, and the number of available beds. The output variables included the average length of stay and the bed turnover interval. The results show that technical efficiency was lower than scale efficiency, and the regression analysis confirmed a statistically important relationship between the variables of the average length of stay and the number of available beds.
Likewise, Ghahremanloo et al. [bib_ref] A novel DEA model for hospital performance evaluation based on the measurement..., Ghahremanloo [/bib_ref] point to the importance of performance evaluation as a relevant tool for hospital management. They attempted to develop the traditional DEA Model with new elements that would, in addition to identifying efficient and inefficient units, allow for the introduction of options for changing the input and output indicators, so that the units would become efficient. The proposed new DEA Model includes the evaluation of the overall hospital efficiency. The model's input indicators comprise the number of healthcare professionals, the number of other staff, and the number of beds. The output indicators include the bed occupancy rate and the bed turnover rate. The authors believe the efficiency-and efficacy-based performance evaluation may aid the managers in assessing whether the organization meets its targets, thus facilitating the decision-making process in the area of human resources as well as safeguarding the hospital's production.
The international evaluation of hospitals was addressed by Varabyova et al. [bib_ref] Comparing the Efficiency of Hospitals in Italy and Germany: Nonparametric Conditional Approach..., Varabyova [/bib_ref]. Their research was focused on applying the advanced non-parametric methods (such as DEA and FDH) to evaluate certain Italian and German hospitals. Once again, their input indicators were the number of beds, the number of physicians and the number of nurses (the personnel data are specified in a full-time equivalent), while the output indicators were the selected inpatient adjusted and day cases. The hospital performance evaluation results were interpreted according to the number of beds and the hospital's ownership structure (public/private). It was concluded that the main differences in the hospital degree of efficiency were found in hospitals which differed in the range of medical branches offered, i.e., in their specialization.
Kocisova, K. et al. [bib_ref] Use of the DEA method to verify the performance model for hospitals, Kocisova [/bib_ref] used the Data Envelopment Analysis Model to compare the degree of efficiency of Polish hospitals in the meso perspective. This means that indicators selected for the evaluation of hospital efficiency were aggregated on the regional level (Polish provinces). The results were interpreted on the individual provinces. It was found that 11 of the sixteen provinces were inefficient. The authors then specified an efficient province, one that became a specimen for the inefficient units. They also examined samples of hospitals aggregated in the given province.
An evaluation of 22 hospitals in Spain was performed by Caballer-Tarazona [bib_ref] A model to measure the efficiency of hospital performance, Caballer-Tarazona [/bib_ref]. The authors state that the evaluation of hospital efficiency is of the utmost importance because large amounts of public resources flow into the public health system which should be spent efficiently. The evaluation was, again, performed using the DEA Method with defined inputs (number of physicians, number of beds) and outputs (number of consultations, number of treatments, number of surgeries).
Khushalani, Yasar [bib_ref] Are hospitals producing quality care efficiently? An analysis using Dynamic Network Data..., Khushalani [/bib_ref] inquired into the efficiency of production in hospitals between 2009 and 2013 using the Dynamic Data Envelopment Analysis with regard to the nature of the individual hospitals (healthcare comprehensiveness). The healthcare quality factor was incorporated in the modeling. Using the Pearson's correlation coefficient, the authors confirmed the relationship between the efficiency of quality production and the efficiency of health and surgical care production. They concluded that municipal and university hospitals were less likely to increase their efficiency of quality production.
A study published by Kohl et al. [bib_ref] The use of Data Envelopment Analysis (DEA) in healthcare with a focus..., Kohl [/bib_ref] reviewed 262 articles that applied the DEA Method in public health, with a special focus on the hospital sector. Research publications were classified in clusters according to the purpose of analysis-pure DEA efficiency analysis, developments or applications of new methodologies, specific management questions, i.e., analyzing the effects of managerial specification, such as ownership, on hospital efficiency and a survey on the effects of reforms, i.e., researching the impact of policy making, such as reforms of health systems, on hospital efficiency. In conclusion, the authors assessed possible the pitfalls when performing analyses using the DEA method, and specified the recommendations for the application of the DEA method as a tool which the architects of health policies as well as the hospital managers should use when making decisions on ensuring the hospital's production.
# Research methodology
The modeling of the technical efficiency of hospital human resources was implemented according to the DEA Model, distinguishing between the constant and variable returns to scale. The evaluation of hospital efficiency was performed by transforming the inputs to outputs in relation to other units of the given set. The outputs had a maximization nature, while the inputs had a minimization nature. The first DEA Model was formulated in the study published by Charnes, Cooper, Rhodes. This model is based on the assumption of constant returns to scale and maximizes the efficiency of the evaluated production unit under the condition that the efficiency of all other units is less than or equal to one. Models with variable returns to scale were first published in an article of Barker et al.. The prerequisite of variable returns to scale is the fact that an increase or decrease in the inputs does not lead to a proportional increase or decrease in the outputs. In the case of variable returns to scale, the efficiency of the production unit is higher (or, more precisely, is not lower) than the efficiency in the case of constant returns to scale.
The modeling of technical efficiency was performed using the input-oriented model that expects that inefficient units should reduce their inputs with respect to the outputs attained. However, it is also a well-known fact that reductions in key human resources (physicians, general nurses, and midwives) has a negative impact on the quality of the services provided in both public health and social services. Therefore, inefficient hospitals will not be advised to reduce the number of physicians and general nurses. The results attained will be used to compare the efficiency of a hospital's personnel structure taking into account the size of the hospitals, which, as specified above, are administratively merged, thus creating abnormally large entities (consortia).
The description of the production units evaluated-public hospitals, the inputs and outputs, evaluation models, and the basic outline of the input-oriented DEA Model with constant returns to scale and variable returns to scale-is specified below.
The attained results of the calculation of technical efficiency according to the DEA (using the DEA Frontier Add-In for Microsoft Excel (Microsoft Corp, Redmont, WA, USA)) were analyzed using standard statistical methods, especially correlation (Pearson's corre-lation coefficient) and regression (simple regression). Moreover, the Bootstrap was used, similarly to other studies [bib_ref] How efficient are Greek hospitals? A case study using a double bootstrap..., Kounetas [/bib_ref]. Statistical analyses were performed using the IBM SPSS software (IBM, Armonk, NY, USA). Bootstrapping is a computer technique that understands a sample as a basic file, from which the computer generates new sample files (so-called Bootstrap samples), having the same size as the original file. From the above procedure, the standard deviation can be estimated, which is used to calculate the confidence interval.
## Production units evaluated in the context of the health system
As of 31 December 2019, a total of 194 public and private hospitals operated in the CR, with the total capacity of 60,633 beds. A hospital fulfils a number of functions. Its basic function is providing diagnostic and therapeutic services and other supporting activities which predominantly fall within secondary and tertiary healthcare. From the functional viewpoint, hospitals may be subdivided into several categories. Primarily, hospitals may be subdivided, according to the health services they provide, into university hospitals, hospitals that provide acute inpatient care, and aftercare hospitals focusing on aftercare and long-term care. In terms of acute inpatient care valuation, it is suitable to categorize hospitals according to their size (the number of beds) and comprehensiveness of care, and more specifically into the following groups: university hospitals and big regional hospitals; healthcare facilities providing highly specialized care; regional and provincial hospitals providing comprehensive care; and provincial hospitals with lower comprehensiveness of care. Other commonly used typologies of hospitals within research areas include the categorization according to the average treatment period; promoters/founders (ministry, region, municipality, natural person, church organization, other legal entity); type of ownership of healthcare facilities; or the ratio between the number of specialized healthcare professionals and other employees.
Hospitals provide acute inpatient and outpatient care and belong to the pillars of healthcare, together with specialized outpatient clinics and health centers. According to the Czech Statistical Office [24], 40.25% of all expenses for public health were spent between 2010 and 2018 in the Czech Republic for inpatient and outpatient care. The hospital network comprises both public and private hospitals, while public hospitals are unambiguously dominant. It must be added in this regard that after 1989, the Czech public health system was hugely affected by political, economic, and social changes, strongly accentuating liberalization, deconcentration, and decentralization. Significant changes implemented in the last 30 years were aimed at making the public health system more efficient, whether from the perspective of the operation of the hospitals or in terms of changes in the hospital care funding. The transformation was related to the changes in hospital promoters, as well as to the changes in the owners of hospital assets. This brought a large difference of opinions, not just in the political arena, but also with the public, who feared problems with the real availability of the hospital by car [bib_ref] Úhrady nemocniční péče vČR, Háva [/bib_ref]. In addition, Nuti et al. [bib_ref] The Challenges of Hospitals' Planning & Control Systems: The Path toward Public..., Nuti [/bib_ref] point out that many of the changes that are being made in the healthcare segment are based on both external factors and the development of the organizational framework for healthcare.
This research focuses on public hospitals that provide comprehensive acute inpatient care. This means that the individual hospitals provide inpatient care in at least three of seven basic medical specialties (internal medicine; surgery; pediatrics; gynecology and obstetrics; anesthesiology and intensive care medicine; neonatology; neurology). Public hospitals are those administered (contributory organizations) or established (public limited companies) by public authorities.
The set of 47 public hospitals investigated was assessed as a whole. The efficiency results were also reviewed from the hospital size perspective. The hospital size criterion chosen was the number of beds, with the resulting three hospital sizes:
- 26 hospitals with a number of beds <499-indicated as Small (S); - 13 hospitals with a number of beds >500 and <999-indicated as Medium (M); - 8 hospitals with a number of beds >1000-indicated as Big (B).
## Inputs and outputs
Inputs and outputs were evaluated, the values of which corresponded to the average value for three years, 2017-2019. The three-year average was chosen intentionally, for two reasons. The first reason was the fact that results for one year may be misleading in some hospitals (e.g., due to investments or extraordinary events); the second was the need to cover the most current staffing and performance condition of the hospitals before the pandemic [fig_ref] Figure 1: Distribution of the inefficiency rate in the models monitored [/fig_ref] , i.e., the year 2019.
Three input indicators were chosen to fulfil the objective of the article-the number of physicians, the number of nurses, and the number of other staff. An optimal ratio between the healthcare professionals and non-medical staff is crucial for the efficient operation of a hospital. It must be noted that this ratio cannot be the same in each healthcare facility because it mainly depends on the proportional design of the hospital and whether the hospital itself ensures all necessary activities or outsources certain services. In the Czech Republic, hospital staffing is specified in two laws: in Act No. 95/2004 Coll., on the requirements for the acquisition of recognition of professional competence to practice the profession of physician, dentist, and pharmacist, as amended [bib_ref] Requirements for Acquisition of Recognition of Professional Competence to Practise the Profession..., Act [/bib_ref] ; and Act No. 96/2004 Coll., on the conditions for attaining and recognizing qualifications to perform professions other than medical professions and to perform activities relating to health care provision and on the amendment to some other acts, as amended. The data structure is based on the requirements for human resources in the individual healthcare wards in accordance with Decree No. 99/2012 Coll., on the minimum staffing requirements for health services. Indicators regarding the number of employees are based on the converted average annual number of registered employees and contractual workers.
The output indicators included the number of outpatient treatments, the number of hospitalized patients, and the number of surgeries. For the purpose of the analysis, data were taken from the annual reports of the individual hospitals for the periods in view, as well as from the National Register of Hospitalized Patients, the National Register of Healthcare Providers, and the National Register of Healthcare Professionals. The data were always valid as of the last day of the given calendar year. The indicators were chosen so that the condition of data availability and relevance was fulfilled in order to ensure clear identification, and so that the sample size and data quality did not significantly decrease the informative value and data comparison. As specified above, data for three years were investigated, in particular to minimize the influence of random, unrepeated fluctuation in the hospital production. [fig_ref] Table 1: Input and output indicators [/fig_ref] below specifies the input and output indicators, including the detailed characteristics. [fig_ref] Table 2: Input and output statistics [/fig_ref] , below, characterizes the statistics of input and output indicators. The table makes it clear that the set of 47 public hospitals comprises groups of hospitals with substantially different sizes (according to the number of beds), which affects the defined indicators. The healthcare staffing was determined according to the expertise of the individual healthcare professionals, as well as the kind and specialty of healthcare. Within acute inpatient care, the workload of medical staff was established according to the number of beds in the given ward (see Act No. 99/2012 Coll.). The table shows that the largest values are attained by state-owned university hospitals-significant backbone hospitals with highly specialized healthcare and the most comprehensive provision of health services. Six out of 10 university hospitals were included in the set. On the contrary, the lowest values were attained by provincial hospitals which comply with the methodical criteria for entry in the hospital sample and perform surgeries within the individual specialties. t
## Models
The staff-performance estimation of efficiency according to the DEA Model is implemented using eight specific models. The first two models contain all three inputs and outputs and estimate the efficiency with constant returns to scale (CRS) and the efficiency with variable returns to scale (VRS). These models are indicated as total (T). Consequently, the partial efficiency is estimated from the perspective of the individual inputs (x1, x2, x3) compared to all outputs, in terms of both the CRS and VRS-see [fig_ref] Table 3: Hospital efficiency models [/fig_ref].
## Data envelopment analysis model: input-oriented models
The Data Envelopment Analysis Model is a non-parametric method widely used for the evaluation of relative efficiency and performance of a set of decision-making units (DMUs). It is based on the maximization of the weighted sum of outputs produced by the unit evaluated divided by the weighted sum of inputs of the same unit, while assuming this ratio must be less than or equal to 1 for all other units. Unit homogeneity is an important prerequisite for the use of the DEA Models.
The DEA Model allows for the discrimination between input and output orientation during the calculation. This article only uses input-oriented models. Furthermore, the DEA Model discriminates the returns to scale, especially the constant returns to scale (CRS), which refer to the overall efficiency, and the variable returns that estimate the net efficiency.
The breakdown of technical efficiency, calculated as the quotient of CRS and VRS, also allows for the determination of the scale's efficiency.
The following is a mathematical formulation of the input-oriented DEA Model under the conditions of constant returns to scale:
[formula] maximize eff U q = ∑ r k u k y kq ∑ m i v i x iq , under the conditions ∑ r k u k y kj ∑ m i v i x jk ≤ 1, j = 1, 2, . . . , n, u k ≥ ε, k = 1, 2, . . . , r, v i ≥ ε, i = 1, 2, . . . , m,(1) [/formula]
where the unit efficiency U q is expressed by eff(U q ), ε is a constant ensuing the condition of positiveness of weights of the inputs and outputs, x ij , i = 1, 2, . . . , m, j = 1, 2, . . . , n indicates the value of the i-th input for the unit U j , and y kj , k = 1, 2, . . . , r, j = 1, 2, . . . , n indicates the value of the i-th input for the unit U j . The calculation of the input-oriented DEA Model with variable returns to scale may be expressed as follows:
[formula] maximize θ U q = ∑ r k u k y kq +µ, under the conditions ∑ r k u k y kj +µ ≤ ∑ m i v i x ij , j = 1, 2, . . . , n, ∑ m i v i x iq = 1, u k ≥ ε, i = 1, 2, . . . , r, v i ≥ ε, j = 1, 2, . . . , m, µ-free. (2) [/formula]
The interpretation of results for the individual units is analogous to the CCR Modelthe efficient unit θ(U q ) equals 1, while θ(U q ) < 1 applies to efficient non-efficient units.
# Results
## Total and partial models
The results of the hospital efficiency calculations according to the input-oriented CRS and VRS models show that hospitals (DMUs) are less efficient in the CRS models, as expected, compared to the VRS models. However, the differences between the models that discriminate between the returns to scale are not large, averaging 8%. The above is confirmed by the number of fully efficient (e = 1, 100%) hospitals; the T_CRS model comprises seven fully efficient hospitals and the T_VRS model has 18 fully efficient hospitals. It is further true that if a hospital is fully efficient in the CRS models, it is also fully efficient in the VRS models.
The average results of partial models focusing on the individual inputs (x1-physicians, x2-nurses, and x3-other staff) in relation to all results (y1-number of hospitalized patients, y2-number of outpatient examinations, y3-number of surgeries) show that x1 (physicians) is the least efficient input in terms of both the constant and variable returns to scale. Input x3 (other staff) shows the second-lowest average results. Input x2 (nurses) attains the best results on average. Inefficient hospitals, with the efficiency level below the average efficiency (CRS 83% and VRS 91%) should seriously consider reducing some of the inputs or increasing some procedures-outputs. [fig_ref] Table 4: Average efficiency values [/fig_ref] shows the average values in the models of the overall technical efficiency CRS and net technical efficiency VRS including the 95% confidence interval, which expresses the upper and lower limits of the average values of efficiency according to the Bootstrap. The confidence interval was also used to specify the inefficiency rate (IR), as follows:
- mild inefficiency: 1> IR ≥ Upper; - moderate inefficiency: Upper > IR ≥ Lower; - strong inefficiency: IR < Lower.
The distribution of results between the individual inefficiency levels (mild, moderate, strong) in the individual models is affected by the orientation of the returns to scale model, see [fig_ref] Figure 1: Distribution of the inefficiency rate in the models monitored [/fig_ref]. In the CRS models, inefficiency is divided into the individual levels more proportionately than in the VRS models. The results may also suggest that the VRS models are more likely to tend to the extremes within the result distribution. The partial models confirm that models x1_CRS/VRS attain the worst results, while the best results are attained by x2_CRS/VRS. The results of the correlation analysis using the Pearson's correlation coefficient are shown in [fig_ref] Table 5: Correlation matrix. [/fig_ref]. The correlation coefficients monitor whether the increase or decrease of a value of one attribute causes the increase or decrease of another attribute. The correlation analysis attributes are the results of the total as well as partial models of efficiency calculations. The correlation matrix shows that there is a positive dependence between the individual attributes (an increase of one attribute leads to an increase of another attribute), which has been verified on the level of significance α = 0.001. Logically, the strongest correlation is between the attributes outside the individual models, CRS and VRS. However, correlation was also documented between the attributes across the CRS and VRS models. The lowest intensity of reaction to the growth of the value of the total efficiency models (T_CRS and T_VRS) was detected in the partial models x1_CRS and x1_VRS, i.e., those oriented to the evaluation of efficiency of the physicians. On the contrary, the strongest reaction to the growth of the efficiency value was reported in the partial models x3_CRS and x3_VRS at T_CRS and partial models x2_CRS and x2_VRS at T_VRS.
## Models according to hospital size
The set of 47 hospitals in view was divided into three groups with different sizes , to discriminate the efficiency results with respect to hospital size.
The subdivision of the efficiency calculation results in terms of hospital size confirms that the highest number of fully efficient hospitals belongs to the category of small hospitals; on the contrary, the lowest number of fully efficient hospitals belongs to the category of big hospitals. Big hospitals, especially university hospitals, are the least efficient within the set in view. Only three big hospitals attain full efficiency in the VRS models, the best of them being H29, which is fully efficient in both the CRS and VRS models. The most balanced are attained by medium-sized hospitals-see [fig_ref] Table 6: Efficiency results according to the hospital size groups [/fig_ref]. Partial models are represented graphically using the regression models (T_CRS and T_VRS is the dependent variable), confirming the results specified in [fig_ref] Table 6: Efficiency results according to the hospital size groups [/fig_ref] , including the fact that the worst results are attained in the partial model focused on x1, i.e., physicians. The reason for determining the dependent variables in the form of T_CRS and T_VRS is that the partial models can be used to explain and predict the value of the dependent variable, not the other way around.
The charts shown below [fig_ref] Figure 2: Results within the regression analysis context [/fig_ref] make it clear that medium-sized hospitals attain the most balanced results. Big hospitals are the least efficient group of hospitals. Small hospitals attained the best as well as the worst results. Comparison of the CRS and VRS models (e.g., a and b) shows that in the CRS models, the results tend to accumulate toward the average in a larger extent compared to the VRS models. Regression models also show that the partial models x2_CRS, x2_VRS have the most significant positive effect on the growth of the value of the overall models. The x1_CRS, x3_CRS, x1_VRS and x3_VRS models have a comparable effect on the dependent variables T_CRS and T_VRS.
# Discussion
The analysis of a sample of 47 public hospitals made it clear that the differences in the technical efficiency of public hospitals were caused by the varying, but not very dissimilar efficiency of the individual type of human resources (inputs)-physicians, nurses, and other staff. The results of technical efficiency were affected by the hospital size (the number of beds) as well as the returns to scale.
The least efficient human resource in relation to the outputs in view (the number of hospitalized patients, the number of outpatient examinations, and the number of surgeries) is the input physicians, as the average efficiency of this inputs is 8-12% lower than the total average technical efficiency values, which range between 91% and 47%. The most numerous human resource in the hospitals is other staff; the average efficiency of this input is comparable to, or no more than 9% lower than, the total average technical efficiency values. The third human resource-nurses-is the least inefficient human resource, as its average technical efficiency value is no more than 4% lower than the total average technical efficiency values. Likewise, a finer resolution of the results according to the inefficiency level (mild, moderate, strong) confirms the above findings, although a varying distribution of inefficiency in the CRS and VRS models may be found. These results support the opinion that the CRS models are more accurate in this estimate of technical efficiency, when limiting the chosen inputs and outputs and the specific set of hospital DMUs, and they are better in revealing inefficiency in both the total and partial representation.
The above results may indicate-within the context of the evaluation logic according to the input-oriented DEA Model-that inefficient hospitals should primarily consider reducing the number of physicians and other medical staff. However, this recommendation must be considered individually in the conditions of particular hospitals, especially those showing an extensive inefficiency degree, even if this includes at least 31% of the hospitals in view.
The results also suggest that the initial hypothesis-that the least efficient input is other staff-was not correct.
Inefficiency in the hospitals in view depends on their size, as documented by the technical efficiency results when divided into three size groups in the hospitals in view. Big hospitals (university hospitals and consortia), although least represented in the set in view (eight hospitals), are the least efficient, and their results lie under the average of the technical efficiency value of the whole set, by at least 15%. Small and medium-sized hospitals show comparable results within the evaluation, while medium-sized hospitals are slightly better. In terms of hospital size and specific input, the results are identical to the above findings, as shown through regression analysis, proving that the type of inputs affects the overall technical efficiency results.
The evaluation of the technical efficiency results according to hospital size shows that big hospitals are not the leaders within the set in view-on the contrary, they are the least efficient units of the set in view. The initial hypothesis about the returns to scale is not true if human resources in public health are the factor of savings. In this regard, the connection between the hospital size and the subsequent, almost monopolistic position in the region is the decisive factor, and the basis for the occurrence of X-inefficiency.
The evaluation of technical efficiency and the results must also be perceived in a wider context, including regional differences as well as the original healthcare system of the given country, as mentioned by other authors [bib_ref] The effectiveness of public investment in human capital, Kozuń-Cieślak [/bib_ref] [bib_ref] The use of Data Envelopment Analysis (DEA) in healthcare with a focus..., Kohl [/bib_ref] inquiring into the evaluation of technical efficiency of hospitals according to the DEA Model. The results are therefore consistent with the report of the OECD, which states that better healthcare leads to a longer life expectancy in the Czech Republic in both men and women, and to a healthier aging process. On the one hand, the Czech Republic is a leader among the Central and Eastern European countries as regards health results; but, on the other hand, the country wrestles with various systemic encumbrances and regional differences which decrease healthcare efficiency.
Based on this research, two problem areas were identified which should be considered by public policy makers on both the national and regional level. This applies to the support in optimizing the structure of human resources in public hospitals. In this regard, it seems advisable to increase the number of nurses while rationalizing the number of other staff, with respect to the special requirements of therapeutic and diagnostic processes in the individual hospitals. In the case of physicians, any interference in their numbers should only be made based on special evaluation processes, because a reduction in the number of physicians is likely to decrease the quality of healthcare [bib_ref] Human resource management and performance in healthcare organisations, Harris [/bib_ref] [bib_ref] Antecedents and Consequences of Corporatization: An Empirical Analysis of German Public Hospitals, Lindlbauer [/bib_ref].
# Conclusions
In conclusion, it is worth noting that the process of selecting input and output variables for modeling technical efficiency through the DEA model changes the process of analysis, which acquires objective characteristics and limits subjectivity in measuring these effects. The suitability of the selection of variables is always determined with regard to the objective of the research while respecting the basic rules of construction of DEA models. This also brings a certain limitation to this model, and therefore the results of the analysis cannot be generalized.
[fig] Figure 1: Distribution of the inefficiency rate in the models monitored. CRS: constant returns to scale; VRS: variable returns to scale. T: total efficiency; x1-physicians; x2-nurses; x3-other staff; y1-number of hospitalized patients; y2-number of outpatient examinations; y3-number of surgeries. [/fig]
[fig] Figure 2: Results within the regression analysis context. (a) Regression line T_CRS and x1_CRS. (b) Regression line T_VRS and x1_VRS. (c) Regression line T_CRS and x2_CRS. (d) Regression line T_VRS and x2_VRS. (e) Regression line T_CRS and x3_CRS. (f) Regression line T_VRS and x3_VRS. Note: S = Small hospitals; M = Medium hospitals; B = Big hospitals. [/fig]
[fig] Author: Contributions: Data curation, I.V. (Ivana Vaňková); Methodology, I.V. (Iveta Vrabková); Writing-original draft, I.V. (Iveta Vrabková) and I.V. (Ivana Vaňková); Writing-review and editing, I.V. (Iveta Vrabková) and I.V. (Ivana Vaňková). All authors have read and agreed to the published version of the manuscript. [/fig]
[fig] Funding: This paper was supported by project SGS No. SP2021/67, International Evaluation of the Performance of Health and Social Services with a Focus on the Application of Multicriteria Methods, Faculty of Economics, VSB-Technical University of Ostrava. [/fig]
[table] Table 1: Input and output indicators. [/table]
[table] Table 2: Input and output statistics.Note: x1-physicians; x2-nurses; x3-other staff; y1-number of hospitalized patients; y2-number of outpatient examinations; y3-number of surgeries. [/table]
[table] Table 3: Hospital efficiency models. [/table]
[table] Table 4: Average efficiency values. [/table]
[table] Table 5: Correlation matrix. [/table]
[table] Table 6: Efficiency results according to the hospital size groups. [/table]
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The formation of ordered nanoclusters controls cadherin anchoring to actin and cell–cell contact fluidity
## Video 2.
Actin retrograde flow in a cis-Ecad-GFP expressing cell spread on Ecad-Fc. A431D cells expressing Life-Act Ruby and cis-Ecad-GFP were allowed to spread on Ecad-Fc substrate for 2 h. Images were analyzed by time-lapse confocal microscope equipped with a Nipkov disk. Time-lapse images were acquired using an inverted microscope (DMI4000; Leica; equipped with a 63× objective) coupled to a spinning-disk confocal module (CS20 head; Yokogawa Electric Corporation) and an intensified CCD camera (Quantem S12SC; Roper Scientific), driven by MetaMorph software. Frames were taken every 500 ms for 3 min. The total duration of the movie is 180 s.
## Video 3.
Ecad-Fc magnetic bead displacement under force (wt Ecad-GFP expressing cells). Ecad-Fc-coated beads were allowed to bind to wt Ecad-GFP expressing cell for 1 h. Force was applied from the sixth frame and maintained until the end of the movie, from left to right. Images were acquired on a microscope (DMIRB; Leica; 100× oil objective) equipped with a CCD camera (Coolsnap HQ2; Roper Scientific). Frames were taken in the burst mode (∼13 frames/s) for 170 s.
## Video 4.
Ecad-Fc magnetic bead displacement under force (cis-Ecad-GFP expressing cells). Ecad-Fc-coated beads were allowed to bind to cis-Ecad-GFP expressing cell for 1 h. Force was applied from the sixth frame and maintained until the end of the movie, from left to right. Images were acquired on a microscope (DMIRB; Leica; 100× oil objective) equipped with a CCD camera (Coolsnap HQ2; Roper Scientific). Frames were taken in the burst mode (∼13 frames/s) for 170 s.
[fig] Figure S2: Nanometric organization of wt and cis-Ecad-GFP at the cell membrane. (A) Whisker plot showing the mean number of NPs per squared micrometer for all images acquired for wt and cis-Ecad expressing cells. Number of images (n) is given in the top of each chart box. (B) Similar representation showing the mean number of NPs per squared micrometer for the images used in the analysis shown in Fig. 2 (all images have from 2 to 100 NPs per picture; 4-180 NP/µm 2 ). A mean of 47.2 and 45.8 NP/µm 2 was found for wt and cis-Ecad expressing cells, respectively. (C) An additional example of clusters seen on the field analyzed for wt Ecad as in Fig. 2 (4 to 180 NP/µm 2 ). Images are 100 × 100 nm. Bars, 15 nm. (D) TEM visualization of high density labeling for plasma membrane sheets of wt Ecad-GFP. Large images are 742.7 × 742.7 nm; Bars, 100 nm. Number of NPs in the picture is given at the bottom left. (top right) Zoom-in of the previous large-scale image at 100 × 200 nm. Bar, 15 nm. (bottom) Insets at 200 × 200 nm. Bars, 15 nm. [/fig]
[fig] Figure S4: Single cell migration of A431D-transfected cells. wt Ecad and cis-Ecad expressing cells were seeded at low density on fibronectin-coated surfaces, imaged, and individually tracked over time. (A) 120-min trajectories of single wt Ecad (left) and cis-Ecad (right) expressing cells migrating on fibronectin (n ≥ 22 cells). (B) Mean length (± SEM) of the trajectories. [/fig]
[fig] Figure S5: Division rate of A431D-transfected cells. (A) Bar graph showing the number of mitotic figures counted on phase-contrast images of wt Ecad and cis-Ecad monolayers during 6 h (six videos analyzed for each condition). (B) Subconcluent wt Ecad and cis-Ecad cell cultures were subject to a 1-h 5-ethynyl-2-deoxyuridine (Edu) pulse and the proportion of Edu + wt Ecad and cis-Ecad cells evaluated. Results are given as mean ± SEM. [/fig]
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Biphasic activation of complement and fibrinolysis during the human nasal allergic response
## Biphasic activation of complement and fibrinolysis during the human nasal allergic response
To the Editor:
The interlinked pathways of complement activation, coagulation, fibrinolysis, and fibrosis contribute to numerous respiratory diseases. Multiple proteases, including mast cell tryptase released in the allergic reaction, have been documented to activate complement. In an extensive gene expression analysis, we recently observed changes in complement factors in mucosal curettage samples following nasal allergen challenge (NAC). Although complement activation has been reported in response to allergen, it has hitherto not been characterized as a detailed in vivo kinetic response. We therefore investigated the kinetics of allergen-induced activation of the complement and coagulation cascades in nasal mucosal lining fluid.
The NAC protocol, demographic characteristics, and clinical response to challenge are summarized in the Results section and [fig_ref] FIG 1: Mast cell degranulation, type II inflammation, complement activation, and fibrinolysis following NAC [/fig_ref] in this article's Online Repository at www.jacionline.org. Two phases of the allergic response have been described: the early allergic reaction (EAR, between 0 and 2 hours post-NAC) and the late allergic reaction (LAR, >2 hours post-NAC). Levels of prostaglandin-D2 (PGD2) rose rapidly, peaking at 5 minutes in the EAR and returned to baseline by 1 hour post-NAC [fig_ref] FIG 1: Mast cell degranulation, type II inflammation, complement activation, and fibrinolysis following NAC [/fig_ref] , probably resulting from mast cell activation; these kinetics closely match a previous observation of b-tryptase release by mast cells. Levels of the activated complement components C3a, C4a, and C5a peaked at 30 to 45 minutes, with a second peak observed at 480 minutes [fig_ref] FIG 1: Mast cell degranulation, type II inflammation, complement activation, and fibrinolysis following NAC [/fig_ref]. (MMP9) and the type II mediators IL-5 and IL-9 were significantly elevated in the LAR, peaking at 480 minutes [fig_ref] FIG 1: Mast cell degranulation, type II inflammation, complement activation, and fibrinolysis following NAC [/fig_ref]. Because complement can activate the coagulation cascade, we measured mucosal levels of coagulation factors. We observed high levels of tissue factor (TF) using a TF-dependent thrombin generation assay, but levels were unaltered by NAC (see A, in this article's Online Repository at www.jacionline.org). These data suggested that coagulation can occur in the upper airway without the need for local TF production post-NAC. Levels of intermediates of coagulation were below detection limits (see the Results section).
Coagulation generates fibrin that is cleared by fibrinolysis, resulting in the formation of fibrin degradation products including D-dimer. Strikingly, D-dimer levels also increased in a biphasic manner, reminiscent of complement components, with peaks observed at 45 and 480 minutes after NAC [fig_ref] FIG 1: Mast cell degranulation, type II inflammation, complement activation, and fibrinolysis following NAC [/fig_ref]. Similarly to complement, late-phase D-dimer elevation was evident in only approximately 50% of participants. Fibrinolysis is plasmin-dependent and the plasminogen activation cascade has been associated with asthma and allergies, 1-3 but no significant increases in tissue-type plasminogen activator nor plasminogen activator inhibitor 1 activity were observed (data not shown). In contrast, levels of urokinase-type plasminogen activator (u-PA) increased during the LAR [fig_ref] FIG 1: Mast cell degranulation, type II inflammation, complement activation, and fibrinolysis following NAC [/fig_ref] see [fig_ref] FIG 2: Distinct phases of complement activation and fibrinolysis are associated with the EARs... [/fig_ref] , I, in this article's Online Repository at www.jacionline.org).
A correlation matrix was established to determine the interrelation between markers of mast cell activation, complement activation, type II inflammation, and fibrinolysis during the EAR and LAR. The matrix analyzed area under the curve values of the induction of mediators over the EAR and LAR [fig_ref] FIG 2: Distinct phases of complement activation and fibrinolysis are associated with the EARs... [/fig_ref].
The P values and Spearman R scores of this matrix are listed in [fig_ref] TABLE E1: Correlation matrix significance values [/fig_ref] , respectively, in this article's Online Repository at www.jacionline.org.
Interestingly, EAR PGD2 levels, a measure of mast cell activation, did not correlate with any LAR response, including type II inflammation. However, PGD2 and early C5a levels were significantly associated (P 5 .04; [fig_ref] FIG 2: Distinct phases of complement activation and fibrinolysis are associated with the EARs... [/fig_ref]. Trends were observed between C5a and D-dimer levels, in both the EAR and LAR (P 5 .12 and P 5 .07, respectively). LAR levels of C5a correlated with u-PA and IL-5 (P 5 .0009 and P 5 .007, respectively; [fig_ref] FIG 2: Distinct phases of complement activation and fibrinolysis are associated with the EARs... [/fig_ref]. In addition, a strong correlation was observed between IL-5 and D-dimer (P 5 .003, [fig_ref] FIG 2: Distinct phases of complement activation and fibrinolysis are associated with the EARs... [/fig_ref]. These results suggested that complement activation in the EAR was coupled with mast cell activation, whereas a second wave of complement activation and fibrinolysis in the LAR was associated with u-PA and type II inflammation.
This study details the kinetics of complement activation and fibrinolysis in the upper airway following NAC, extending a previous report of gene expression. Validation of nasosorption and biomarker assessment will be required in patients with allergic rhinitis in the context of natural seasonal exposure. This will establish whether these nasal mucosal biomarkers are useful in diagnosis, stratification, and monitoring in patients with allergic rhinitis. Early complement activation is in line with cleavage by mast cell tryptase, whereas in the LAR, associations between C5a, IL-5, u-PA, and D-dimer suggest that complement activation may be a component of type II inflammation [fig_ref] FIG 2: Distinct phases of complement activation and fibrinolysis are associated with the EARs... [/fig_ref] , where proteases such as MMP9 could activate complement. In asthma, sputum eosinophils have been reported to correlate with D-dimer levels 2 and complement anaphylatoxins have been associated with maturation of maladaptive immune responses. Similarly, extensive complement deposition in nasal polyps of patients with chronic rhinosinusitis is closely associated with local IgM and anti-DNA IgG levels. In addition, the complement and coagulation cascades contribute to idiopathic pulmonary fibrosis. Activation of these pathways may represent a common feature of numerous airways diseases and contribute to components of remodeling and fibrosis. Therefore, analysis of complement, coagulation, and fibrinolysis cascades in airway mucosal lining fluids will be of interest in a range of respiratory diseases. A, Correlation matrix of the induction of each mediator over the early (0-2 hours) and late (3-8 hours) allergic reactions; blank squares denote insignificant (P > .05) correlations, and color denotes Spearman R value. B, Activation of the complement and coagulation cascades in the EAR and LAR is summarized, along with cells and mediators likely to be implicated in these processes. C, Proposed model of the distinct phases of complement activation in the EARs and LARs. In the early phase, mast cell products trigger complement activation, which, in turn, activates TF, which is abundant in the upper airway, resulting in fibrin deposition. Fibrinolysis rapidly follows, resulting in D-dimer formation. In the late phase, complement may be activated by proteases associated with type II inflammation, similarly triggering fibrin deposition through TF activation. In the late phase, u-PA levels rise, resulting in increased plasmin generation from plasminogen. Plasmin contributes to fibrinolysis and D-dimer formation. Identification of clinically relevant chronic rhinosinusitis endotypes using cluster analysis of mucus cytokines
To the Editor: Chronic rhinosinusitis (CRS) pathophysiology has yet to be clearly defined, largely because the diagnosis itself likely represents a heterogeneous syndrome rather than a distinct clinical entity. Several investigators have shown that CRS with nasal polyps (CRSwNP) and CRS without nasal polyps (CRSsNP) can both be linked with T H 1-, T H 2, or T H 17-associated inflammatory signatures, 1,2 and there likewise appears to be a substantial geographic predisposition to select types of inflammatory burden. Recent attempts to identify inflammatory endotypes have presented a more nuanced approach to the classification of CRS, with Tomassen et al 4 recently using surgically obtained tissue to identify potential CRS endotypes based entirely on inflammatory biomarkers. While such studies have preferentially used tissue-based assessment of inflammatory mediators, this approach may have some logistical limitations because it is inherently invasive and subject to variations in site-specific protein expression throughout the sinonasal cavity. Minimally invasive approaches to disease endotyping have been reported for asthma and other respiratory diseases, typically using analysis of sputum, mucus, or epithelial brushings. The aim of the current study was to define CRS endotypes in a US population entirely on the basis of analysis of mucus collected via a minimally invasive approach. Going a step further, we sought to additionally define the clinical relevance of putative
## Methods volunteers, recruitment, ethics
Volunteers with a history of allergic rhinitis during the grass pollen season were recruited. All volunteers were tested for sensitization to timothy grass pollen by allergen skin prick testing, where a positive test result was defined as a wheal with a diameter of more than 3 mm larger than that achieved with negative control. Participants were not using any anti-inflammatory therapies, nor had they used local or systemic corticosteroids in the 2 months before NAC.
The study was approved by the London -Harrow Research Ethics Committee (REC#13/SM/1837). Allergen challenges were performed by administering 5000 SQ-U of Phleum pratense (timothy grass) pollen (ALK Abello, Horsholm, Denmark) per nostril using Bidose nasal delivery devices (Aptar Pharma, Radolfzell am Bodensee, Germany).
## Measurement of total nasal symptom scores and peak nasal inspiratory flow
Total nasal symptom scores were scored by participants on a 12-point scale, with 0 to 3 points given to each of nasal congestion, itching, sneezing, and rhinorrhea (0 5 none, 1 5 mild, 2 5 moderate, 3 5 severe symptoms). Measurements were taken at each sampling time point. Nasal patency was measured by peak nasal inspiratory flow using a Youlten nasal peak flow meter with the highest result of 3 measurements used for analyses.
## Sampling
Nasal lavage was performed using 5 mL normal sterile saline on all volunteers 60 minutes before allergen administration. Nasal fluid was collected using nasosorption devices (Hunt Developments Ltd, Midhurst, UK). Sampling was performed at the inferior turbinate, with absorption performed for 60 seconds. Collections were performed at baseline (15 minutes before allergen administration), then at 5, 15, 30, 45, and 60 minutes postchallenge. After 60 minutes, samples were collected hourly up to 8 hours. After collection samples were returned to their storage tubes and immediately dry-frozen at 2808C until analysis. Fluid was eluted from the nasosorption matrix using 300 mL of Millipore assay buffer (Millipore, Billerica, Mass) and centrifugation at 16000g for 20 minutes, at 48C. Samples for measurement of TF were eluted using the same protocol, with the exception that Tris-buffered saline replaced Millipore assay buffer.
## Mediator measurements
Eluted nasosorption fluid was analyzed by immunoassay for a range of inflammatory cascade components. Plasminogen activator inhibitor 1 was measured by ELISA using duoset antibodies as per manufacturer's instructions (R&D Systems, Minneapolis, Minn). PGD2 levels were measured using a PGD2-MOX ELISA kit (Cayman Chemicals, Ann Arbor, Mich). Activity of tissue-type plasminogen activator was measured using Combi Actibind ELISA kits (Technoclone, Vienna, Austria). Levels of the fibrin degradation product D-dimer were quantified using a HemosIL ACL Acustar D-dimer immunofluorescence assay (Instrumentation Laboratory, Bedford, Mass). A luminex assay was used to measure levels of urokinase (u-PA) (R&D Systems) using a Bioplex 200 analyzer (Bio-Rad, Hercules, Calif). C3a, C4a, and C5a levels were measured using the Luminex system with a cytometric bead array anaphylatoxin panel (BD Bioscience, San Jose, Calif). Levels of IL-5, IL-9, and matrix metalloprotease 9 were measured using MSD V-PLEX kits (MSD, Rockville, Md).
## Measurement of tf using calibrated automated thrombography
TF levels in nasosorption eluates were assessed indirectly in real-time thrombin generation assays by calibrated automated thrombography, using a Fluoroscan Ascent FL plate reader (Thermo Labsystems, Philadeliphia, Pa) and Thrombinoscope software (Synapse B.V., Maastricht, The Netherlands). Thrombin generation was initiated in 80 mL pooled normal plasma in the presence of 4 mM phospholipid vesicles and 16.6 mM CaCl 2 in a total volume of 120 mL. The amount of thrombin formed was monitored using a thrombin-specific fluorogenic substrate, Z-Gly-Arg-AMC-HCl (0.42 mM; Bachem, Bubendorf, Switzerland). The nasosorption eluates were added in a 1:100 final dilution. To inhibit contact activation, corn trypsin inhibitor (Enzyme Research Laboratories, South Bend, Ind) was added to the plasma (40 mg/mL plasma). An inhibitory antibody against TF (1 or 10 nM; Sigma, St-Louis, Mo) was added to show that any thrombin generation detected in the eluted nasal fluids was caused by TF.
## Statistical analyses
Statistical analyses of time-course data were performed in GraphPad Prism v7.00 (GraphPad Software, La Jolla, Calif) using Friedman test with Dunn correction for multiple comparisons, based on the level at each time point relative to that at baseline. All plots are shown as medians with interquartile ranges. Correlation analyses were performed in R (R Foundation for Statistical Computing, Vienna, Austria) using the ''ggcorrplot'' package. All correlations were based on mediator data expressed as fold-induction over baseline of each individual. Statistical testing of these correlation plots used Spearman coefficients.
# Results
Fifteen adult volunteers sensitized to timothy grass pollen were recruited. The mean age of participants was 32.4 years (range, 19-57 years), and 8 participants were men. To enable measurements of TF levels, a second cohort (n 5 4) was recruited, as detailed in the Methods section. In the main study, the average wheal diameter following skin prick testing with timothy grass pollen was 8 mm (range, 5-14 mm). The schedule for NAC and respiratory sampling, performed by nasosorption of mucosal lining fluid, is summarized in [fig_ref] FIG 1: Mast cell degranulation, type II inflammation, complement activation, and fibrinolysis following NAC [/fig_ref] Following NAC, peak nasal inspiratory flow fell rapidly to a nadir in the EAR at 30 to 45 minutes (median, 290 L/min; [fig_ref] FIG 1: Mast cell degranulation, type II inflammation, complement activation, and fibrinolysis following NAC [/fig_ref]. Similarly, total nasal symptom score, which is scored out of 12, rose sharply, peaking at 5 minutes (median, 9; range, 3-11; [fig_ref] FIG 1: Mast cell degranulation, type II inflammation, complement activation, and fibrinolysis following NAC [/fig_ref]. In the LAR, clinical symptoms generally consisted of a mild nasal obstruction. The clinical magnitude of the EAR did not predict the clinical magnitude of the LAR. Furthermore, no association was observed between mediator levels and symptoms in the LAR (data not shown).
For measurement of TF, a second cohort of volunteers was challenged with timothy grass pollen using an identical challenge protocol. The mean age of these volunteers was 32 years (range, 27-39 years); 3 were men. The mean wheal diameter following skin prick testing with timothy grass pollen was 6.4 mm (range, 5.5-10 mm).
Levels of the coagulation cascade intermediates thrombinantithrombin complex and prothrombin fragments 1 and 2 were measured in the main cohort (n 5 15) but were below assay sensitivity (15.6 pg/mL and 0.007 nmol/mL, respectively; data not shown). In addition, no significant changes in plasminogen activator inhibitor 1 levels were observed, though levels were frequently below assay sensitivity (0.31 ng/mL; data not shown). ). Friedman test with Dunn correction for multiple comparisons, based on the level at each time point relative to those at baseline, was used to determine statistical significance (*P < .05, **P < .01, ***P < .001, ****P < .0001). MMP9, Matrix metalloprotease 9.
FIG E3. Tissue Factor (TF) is persistently present in the human airway at baseline and during allergen exposure. A, TF levels in the nose were indirectly determined using a TF-dependent thrombin generation assay, carried out at baseline and for 8 hours following NAC (n 5 4). Data are represented as the median and interquartile range of the subjects, where the thrombin generation of each sample was determined in 3 independent technical replicates. B, To confirm that thrombin generation was TF dependent, thrombin generation was determined in the presence of an inhibitory anti-TF antibody (1 or 10 nM) (n 5 8). The thrombin generation of these 8 samples (20 minutes pre-and post-NAC from 4 donors) was determined as the mean of 2 independent technical replicates. Data are shown as mean 6 SEM of percentage change from the 0 nM control in each sample. Inhibition data were tested for significance by 1-way ANOVA (****P < .0001). P values from the correlation matrix [fig_ref] FIG 2: Distinct phases of complement activation and fibrinolysis are associated with the EARs... [/fig_ref]. Spearman R values from the correlation matrix [fig_ref] FIG 2: Distinct phases of complement activation and fibrinolysis are associated with the EARs... [/fig_ref].
J ALLERGY CLIN IMMUNOL VOLUME 141, NUMBER 5
[fig] FIG 1: Mast cell degranulation, type II inflammation, complement activation, and fibrinolysis following NAC. Following NAC, nasosorption was used to measure the levels of inflammatory mediators: (A) PGD2 in the first hour and the active complement components C3a, C4a, and C5a over the 8-hour time series. In addition, levels of (B) IL-5, IL-9, and MMP9 are shown over 8 hours. C, D-dimer and u-PA levels post-NAC. Data are represented as medians (n 5 15). See Fig E2 for statistical analyses. MMP9, Matrix metalloprotease 9. [/fig]
[fig] FIG 2: Distinct phases of complement activation and fibrinolysis are associated with the EARs and LARs. [/fig]
[fig] FIG E1: Study design and clinical response. A, Clinical events during NAC. Nasal lavage was performed before allergen administration. Nasosorption sampling and measurements for total nasal symptom score (TNSS) and peak nasal inspiratory flow (PNIF) were taken at 5, 15, 30, 45, and 60 minutes post-NAC, then hourly to 480 minutes. Clinical severity of the allergic reaction was measured by (B) PNIF and (C) TNSS at each time point. N 5 15. L, Left; R, right. Data are represented as medians with interquartile ranges. [/fig]
[fig] FIG E2: Mediator levels following NAC. Data from Fig 1 are shown as medians and interquartile ranges. Following NAC, mediators and markers of mast cell degranulation, type II inflammation, complement activation, fibrinolysis, and plasminogen activation were measured: A, PGD2 in the first hour post-NAC. B, IL-5. C, IL-9. The complement components (D) C3a, (E) C4a, and (F) C5a. In addition, levels of (G) D-dimer, (H) MMP9, and (I) urokinase (u-PA) were determined (n 5 15 [/fig]
[table] TABLE E1: Correlation matrix significance values [/table]
[table] TABLE E2: Correlation matrix Spearman coefficients [/table]
|
Measurement of Angstrom to Nanometer Molecular Distances with 19F Nuclear Spins by EPR/ENDOR Spectroscopy
Spectroscopic and biophysical methods for structural determination at atomic resolution are fundamental in studies of biological function. Here we introduce an approach to measure molecular distances in bio-macromolecules using19Fn uclear spins and nitroxide radicals in combination with high-frequency (94 GHz/3.4 T) electron-nuclear double resonance (ENDOR). The small size and large gyromagnetic ratio of the 19 Flabel enables to access distances up to about 1.5 nm with an accuracy of 0.1-1 .The experiment is not limited by the size of the bio-macromolecule.P erformance is illustrated on synthesized fluorinated model compounds as well as spinlabelled RNAd uplexes.T he results demonstrate that our simple but strategic spin-labelling procedure combined with state-of-the-art spectroscopya ccesses ad istance range crucial to elucidate active sites of nucleic acids or proteins in the solution state.Supportinginformation and the ORCID identification number(s) for the author(s) of this article can be found under: https://doi.org/10.
# Introduction
High-resolution structural information is fundamental for understanding the function of biological macromolecules like proteins or nucleic acids.B iophysical methods such as X-ray crystallography,cryo-electron microscopy (EM),or nuclear magnetic resonance (NMR)can provide this information under their specific sample requirements.N evertheless, an integrative approach to structural biology which takes into account data from multiple other sources including smallangle X-ray scattering as well as optical and magnetic resonance spectroscopies is being increasingly appreciated.Forinstance,NMR and fluorescence energy transfer (FRET) can provide unique information in the solution state,addressing the inherent structural flexibility and dynamics of biomolecules.Additionally,N MR in the liquid and solid state (SSNMR) provides atomistic or residue resolution but suffers from an overall low spin sensitivity.Electron paramagnetic resonance (EPR) spectroscopy has the advantage of ahigh spin sensitivity on the order of afew picomoles due to the higher polarization of electron spins that have am uch larger magnetic moment as compared to nuclear spins.This advantage gets even more pronounced at high magnetic fields and frequencies,where also spectral resolution increases.Moreover,E PR is not restricted by the size of the biomacromolecule,h owever,i tr equires the presence of at least one paramagnetic center. In the last decade,E PR-based pulsed dipolar spectroscopyhas emerged as an important tool for structural biology,a si ta llows for measuring dipolar coupling between two paramagnetic centers (typically nitroxide spin labels) separated by % 1.5 up to approximately 8nm, where the distance range can be extended by using either sophisticatedly shaped pulsesor deuterated proteins.At such long distances,t he dipolar coupling depends on the inverse cube of the inter-spin separation. Using advances in data analysis,the method provides the distance distribution of the molecular ensemble and thus reports on conformational distributions from the dynamics of the labelled molecules.I np rinciple,d istances below 1.5 nm could also be measured,however, at such short distances it is inadequate to treat the two coupled electrons as localized, quasi-classical point dipoles.Thus,d istance determination is complicated by quantum effects like spin delocalization or exchange couplings.Here we present an approach that conveys the spinlabelling concept from pulsed dipolar spectroscopy to electron-nuclear double resonance (ENDOR) by replacing one of the nitroxides with an uclearFs pin label to access molecular distances 9 1.5 nm. Thevalue ofFnuclear labels has been recently recognized in the NMR literature,w here the large gyromagnetic ratio ofFa nd its scarcity in biological samples have been exploited to selectively measure couplings between 1 Ha ndForb etween pairs ofFn uclei separated by up to % 20 .Thec ombination of nitroxide andFl abelling on cyanovirin-N allowed for measuring distances of 12-24 using paramagnetic relaxation enhancement (PRE) experiments.While requiring the same labelling strategy,P RE and EPR/ENDOR are complementary to some extent. PRE allows measuring distances in liquid solution with an r À6 distance dependence,p roviding access to average inter-spin distances.E NDOR is typically performed in frozen solution and thus potentially provides access to distance distributions as well as information about the orientation of the electron-spin center.A sa na dvantage, EPR-based methods are not limited by the size of the sample molecule and require am uch smaller amount of sample.A n early example of measuringFh yperfine (hf) couplings by EPR was presented by Wells and Makinen, who used cw ENDOR to measure distances from nitroxides toFnuclei in model compounds.However,their study was hampered by the low magnetic field strengths of only % 0.35 T, which led toFr esonances heavily overlapping with 1 Hr esonances.A possibility to circumvent this issue was presented by Zänker et al.,who employed 31 Pnuclei instead of 19 F.Using Mims' ENDOR experiments,couplings as small as % 33 kHz could be resolved corresponding to ad istance of about 1nm between an itroxide and a 31 Pn ucleus.T he availability of high-field/high-frequencyE PR spectrometers (n EPR 0 94 GHz) meanwhile allows for as ufficient resolution of nuclear frequencies,aswereported in recent publications,and for ag eneral implementation of
# Results and discussion
We synthesized four mono-fluorinated nitroxide model compounds 1-4 by Steglich esterificationand subsequently crystallized them by slow evaporation of saturated solutions of 1-4.Asshown in-d, the inter-spin distances R XRD from the X-ray structures range from 6.8 for 1 to 14.8 for 4,using the midpoint of the N À Obond as areference for the electron-spin localization.Echo-detected EPR spectra at 94 GHz of 1-4 showed aline shape typical for nitroxides,with ar epresentative spectrum displayed in.D etails on the syntheses,X -ray structures,9 4GHz EPR experimental setup and experimental parameters are given in the Supporting Information.
Assuming al ocalized electron-spin density,t he hf coupling as af unction of the distance between an itroxide spin label andFc an be estimated using the point-dipole model [Eq. (1)]T
[formula] ¼ m 0 4ph g e g n m B m N = R 3 ¼ C = R 3ð1Þ [/formula]
where T is the principal-axes value (T ? )ofthe dipolar part of the hf tensor, m 0 the vacuum permeability, h the Planck constant, and C = 74.52 MHz 3 is calculated from the g factors and Bohr magnetons of nitroxides (g iso % 2.005) andF, respectively.For distances > 5 ,asinmodel systems 1-4, all expected couplings are smaller than 1MHz. To detect such small couplings,w ec hoose the Mims ENDOR experiment, which has notably highest sensitivity,however, it produces blind spots in the hf spectrum according to the functionF ¼ 0:
[formula] 5sin 2 2p n HFC 2 tð2Þ [/formula]
where F represents the ENDOR efficiency, t is the separation between the two 908 8 preparation pulses,a nd n HFC the expected coupling frequency. Thus,t he t values have to be optimized for each sample to detect the canonical resonances of the dipolar powder pattern (Pake pattern), with principal axes frequencies at n HFC =AE T and AE T/2. Under consideration of the relaxation times T m, Supporting Information), t values were chosen that optimize detection of the parallel component of the dipolar tensor at n HFC =AE T.isplays representative 19 F-Mims ENDOR spectra at 94 GHz for 1 and 4 in [D 6 ]DMSO/[D 4 ]MeOD (1:1.5) at 50 K. Thesample volume amounted to 2 mLatconcentrations of 250-300 mm.A ll individual ENDOR spectra report orientation selectiondue to the narrow excitation bandwidth of the microwave (mw) pulses with respect to the broad EPR line.Inprinciple,the full Pake pattern might be reconstructed by summing over all orientations,which would correspond to measuring and summing spectra at as et of narrowly spaced resonance fields.H owever,t his procedure is experimentally not feasible due to the long spectral averaging,a nd also not necessary.I no rder to find the principal resonance components of the Pake pattern and to optimize experimental time, spectra at only af ew resonance positions A-E, including the canonical orientations of the g tensor, were recorded. As um spectrum is reported for aq ualitative comparison with acalculated, ideal Pake pattern. Summation was performed after normalizing the individual spectra (A-E) to the number of scans.
At position A, high selectivity for the parallel component of the dipolar coupling tensor is observed (for all compounds , and 4 (d) as determined by crystallography.I nter-spin distances R XRD between the midpoint of the NÀObond andFare indicated. Ellipsoids at 50 % probability.C olor code:g ray = C, blue = N, red = O, yellow = F. e) Representative echo-detected EPR spectrum of 1 with ENDOR observer positions A-E. f) Schematic Mims ENDOR pulse sequencedenoting the time intervals t and T. g) Orientation of the nitroxide g tensor axes and definition of the dipolar tensor (R k T x or 2 T)aswell as the Euler angles a and b between g and the dipolar tensor.. This leads to ahigh sensitivity at this observer position, which is even higher than at position B, where the echo intensity is maximized. Owed to this effect, the corresponding features in the sum spectrum are also slightly over-weighted. ENDOR spectra of 2 and 3 looked very similar to those of 1 and are reported in. Inspection of all spectra for 1-3 reveals that peaks appear approximately at half of the frequency of the edges (grid lines) consistent with ad ipolar Pake pattern.
Fors ample 4,t he individual spectra show resonances consistent either with AE T (A) or AE T/2 (B-E). Thes um spectrum, however, does not represent af ully resolved Pake pattern because the resolution no longer suffices,d espite using RF pulses twice as long as for compounds 1-3 (note the factor of 10 in the frequency axis between spectra of 1 and 4). Indeed, aseparation between the spectral features exceeding the ENDOR line width is ar equirement to resolve such features.Inthe case of 4,the line width and the separation of the perpendicular and parallel components are nearly identical, which is why the corresponding spectral features are not as clearly resolved as in the spectra of 1-3 (spectral resolution is discussed further in the Supporting Information). Nevertheless,o wed to the axial symmetry of the dipolar coupling tensor, peaks at AE T/2 are dominant in most of the orientation-selected spectra. Thes implest way to extract inter-spin distances is to read off the T values from the spectraand then apply Equation. Forc ompounds 1-4, the respective values T read are 243 AE 9, 86 AE 6, 63 AE 6, and 21 AE 3kHz, respectively,w here the error was estimated as three times the resolution of the frequency axes.T hese values correspond to distances R read of 6.7 AE 0.1, 9.5 AE 0.2, 10.6 AE 0.3, and 15.2 AE 0.7 ,respectively,inexcellent agreement with the distances R XRD observed in the crystal structure.
To examine the fidelity of as imple point-dipole analysis, we performed spectral simulations by taking into account ac onformational analysis provided by the ORCAq uantum chemistry program package,which allowed finding stable conformers a-d for 1-4.
Fora ll compounds,r otation of the pyrroline-N-oxyl moiety leads to conformation c,which comes at an energetical cost of % 2.5-3 kJ mol À1 when compared to conformation a. Conformation b is energetically equal to conformation a in the case of 2,whereas it is % 1.8 kJ mol À1 less stable than a in the case of 1.T he relative weight W of the conformers was determined based on their respective energies and populations according to aBoltzmann distribution at atemperature of 180 K. While the weighted average of the melting temperatures of DMSO and methanol amounts to % 220 K, we chose this lower temperature to reflect that the solvent system forms ag lass,l eading to decreased freezing temperatures.From the geometry of the conformers we could extract the distances R DFTbetween the midpoint of the NO group and theFnucleus as well as the Euler angles a and b between the nitroxide g tensor and the dipolar tensorand . TheD FT-predicted dipolar couplings T DFT (Supporting Information, Section S4) for all conformers are listed in. Subsequently,all conformers with their respective weights were considered for the ENDOR simulations.hows Mims ENDOR simulations carried out with the software EasySpin.TheD FT-predicted couplings T DFT were slightly re-adjusted to best reproduce the Mims ENDOR spectra, whereas the Euler angles a and b were kept fixed during the simulation. From the simulated T simvalues,the point-dipole distances R sim were computed according to Equation (1). Overall, our approach provided four sets of values for the inter-spin distances,two each from X-ray and DFT-optimized structures R XRD and R DFT ,r espectively,t ob e compared with those obtained from the ENDOR spectra, R read and R sim .W ef ind that the R sim values for the different conformers are consistent with the distances R DFT within about 0.3. Furthermore, R read agrees within its uncertainty with the distance from the conformer that has the highest weight. In the case of 2,w here two high-weight conformers are present, R read is found in between the expected distances for the two conformers.Additionally,the simulation reports al ine-width parameter which contains contributions from different sources.Contributions arising from the experimental settings,s uch as the length of the RF pulse,o rf rom the spin system, such as the nuclear-o re lectron-spin relaxation times T M or T 1 ,are expected to be similar across the samples.Increasing the RF pulse length (that is,decreasing the RF power) consistently led to smaller line widths, however at the expense of sensitivity.F or 1-3,R Fp ulses of % 50 msr epresented ag ood compromise between sensitivity and resolution. In the case of 4,as ignificant improvement of the resolution was obtained by prolonging the RF pulse up to % 100 mswith still sufficient sensitivity (see Supporting Information for spectra of 4 recorded with % 50 msR Fp ulses). Foragiven RF power, the line width decreases from compound 1 to 4, indicating that line widths are also affected by distance distributions.I ndeed, line broadening owed to distance distributions is expected to be the largest for compound 1, where the distances are short. Fore xample,av ariation of AE 0.1 around amean distance of 6.6 leads to aspread in frequencies of % 23 kHz, whereas the same variation around amean distance of 9.6 (2)leads to aspread of only % 5kHz.
[formula] R 2 = F, R 1 = R 3 = H, 3:R 3 = F, R 1 = R 2 = Hand 4:R 1 = p-F-Ph, R 1 = R 2 = H. [/formula]
To examine the method on ab io-macromolecule,t wo 16mer RNAd uplexes RNA1 and RNA2 were designed and investigatedfollowing ap reviously published protocol (see Supporting Information).In both duplexes, strand Ac arries a 19 Fn ucleus at the 2'-position of the ribose attached to an adenine base at position 7( blue in. Strand B( red incarries the semi-rigid TEMPObased C T labelat either position 5(RNA1)orposition 11 (RNA2). Our previous investigations had shown that the C T label allows maintaining base-pairing in the duplex so that an A-RNAc onformation is conserved.To visualize the structures of RNA1 and RNA2,t he coordinates of the 16mer A-RNAh elix with ar aise and at wist of 2.81 and 32.78 8,r espectively,w ere generated by employing the w3dna server.Subsequently,the C T label was introduced in one of the most energetically stable conformations compatible with hydrogen bonding using PyMOL, as predicted by previous DFT calculations of the C T energy surface.TheC T conformations were defined by two dihedral angles f 1 and f 2and values of f 1 % 120 AE 75 and f 2 % 0 AE 60 were found within the energy minimum, with asecond, less broad minimum at f 1 %À60 AE 20 and f 2 % 0 AE 40.These models allow for estimating NO-Fd istances of approximately 12 and 14 in RNA1 and RNA2,r espectively.M ims ENDOR spectra of RNA1 and RNA2 show clearly resolved peaks at AE T (spectrum A) or AE T/2 (spectra B-D), as shown in( RNAs ample preparation, experimental setup,and EPR characterization are detailed inFMims ENDOR spectra of RNA1 and RNA2 (smoothed data in gray, unsmoothed data in black, dotted lines) with simulationsusing asingle C T conformer (red, conformation n for RNA1,conformation b for RNA2). T read is inferred from spectrum C. Distances R read are indicated. Optimized t values were 2200 (RNA1)and 2180 ns (RNA2). RF pulses were 54 ms. The acquisition times amount to approximately 18 and 30 hper spectrum for RNA1 and RNA2,r espectively. Further experimental parameters are given in the SupportingInformation.
the Supporting Information). TheS /N ratio is worse than in the model systems since the sample concentration is about af actor of two lower and the spectra are broader.F rom the individual spectra, the sum spectrum was generated as for 1-4. Thes um spectra do not represent at ypical Pake pattern due to visible broadening,t hus T read was obtained representatively from spectrum C, leading to T read = 43 AE 6kHz and T read = 23 AE 4kHz for RNA1 and RNA2,r espectively.U sing Equation (1), these translate to average 19 F-electron-spin distances of 12.0 AE 0.6 and 14.8 AE 0.9 .
Simulations based on 12 individual conformations with different dihedral angles f 1 and f 2 within the energy minimaindicated that only some conformers with dihedral angles f 1 % 80 AE 40 and f 2 % 15 AE 30 allow for simulating ENDOR spectra that are consistent with the experimental data. Interestingly,t he conformations which agree with the ENDOR data also produce considerably fewer short contacts between the label and the RNA backbone,t hereby providing further evidence that the A-RNAf orm is preserved after labelling with C T .T he best simulation based on asingle conformer is superimposed to the data for RNA1 and RNA2 in. We note that for RNA2,t he spectrum at position Am ight be indicative for as plitting and thus for as econd component. However,t he resolution at the other excitation positions does not allow for as imple,u nambiguous analysis with multiple components. Forb oth RNAd uplexes the T sim values agree within the uncertainty with the read-off values T read. Noteworthy,line-width parameters considerably larger than for 1-4 had to be used to simulate the spectra of RNA1 and RNA2. Furthermore,t he line-width parameter turns out anisotropic and is largest at excitation positions B k g x ,w hich report the 2T (T k )r esonances.T his is consistent with inhomogeneous broadening due to aconformational distribution of inter-spin distances,since the value 2T is affected twice as much by such ad istribution as the T component. Thed etailed analysis (Figures S19-S21) revealed that also ad istribution of Euler angles in the different geometries contribute significantly to the observed ENDOR linewidths in the orientation-selective spectra. It is plausible that the line widths observed for the RNAs reflect ac onformation distribution arising from degrees of freedom of the C T label, including ad istribution of distances on the order AE 0.5 ,c onsistent with previous data derived from PELDOR studies.Figure 5s ummarizes the range of measured dipolar couplings as af unction of the distances.T he plot permits some conclusions about the detection limit. According to the R À3 distance dependence [Eq. (1)], T decreases by afactor of % 10 if the distance increases from 6.7 to % 15 and by an additional factor of approximately 2f rom 1.5 to 2.0 nm. The question arises as to whether ad istance of 2nm( T % 9kHz) would be detectable.Our data show that the resolution of the Mims ENDOR experiment with nitroxides is within the range 10-15 kHz due to the different broadening mechanisms and the spectral hole [Eq.].T he loss of signal intensity due to the spectral hole cannot be compensated by longer t values. Indeed, the optimized t value for 4 is already almost identical to the T M value of the nitroxideand spectral acquisition time amounts to about four days.Therefore,although it might be somewhat sample-dependent, the detection limit at present appears to be 9 1.7 nm. In the case of short distances < 6 ,t he simple point-dipole model might break down, as electron-spin delocalization cannot be neglected anymore.Aplot of the point-dipole model vs. adistributed dipole modelin which aspin-density delocalization between Na nd Oi sc onsidered (Supporting Information, Section S6) already shows this deviation. Additionally,isotropic HFC interactions might occur at such short distances.I nt his regime,t he experiment is still feasible,b ut ad istance interpretation will require quantum-chemical modelling to take into account the detailed spin-density distribution. Forn itroxides in the distance regime studied here,o nly minor improvements are expected by taking into account spin-density delocalization as compared to the simple mid-point analysis,a tt he expense of considerably more modelling. However,w hen studying more delocalized spin centers like aromatic radicals or trityls,adetailed analysis of the spin-density distribution will be necessary.
# Conclusion
In conclusion, using high-frequency9 4GHz EPR/EN-DOR combined with strategicFand nitroxide spin labelling [a] The value in parentheses is the value used for the spectrum at position A. we have reported inter-spin distances up to % 15 in biomacromolecules with accuracies of 0.1-1 that depend on the distances and the sample-specific inhomogeneous broadening. Thea dvantage of this technique as compared to the other widespread approach to measure hyperfine couplings, that is,ESEEM spectroscopy,is the increasing performance towards high field and frequencies,w hich do not rely on the excitation of forbidden transitions and on specific matching conditions.Moreover,ESEEM generally suppresses frequencies corresponding to the canonical orientations,w hich are crucial in this analysis and could only be indirectly obtained from ESEEM data by detailed simulation. Compared to other methods available in structural biology,the advantages are the atomic resolution, the absence of limitations by protein size,a nd sensitivities in the subnanomole range,w hich can complement, for instance,c ryo-EM in frozen-solution studies or access macromolecules not amenable by NMR methods.M oreover,w ide applicability is expected since the methods are already well-established for introducingFl abels into proteins and nucleic acids.E xamples include fluorinated purine and pyrimidine nucleobasesand 2'-fluoro ribose modificationsas well as fluorinated amino acids, particularly when introduced site-specifically into recombinant proteins as artificial amino acids by amber stop-codon suppression.Structural knowledge from distances at atomic resolution plays ac entral role,f or instance,i nm echanistic studies of enzymesand functional nucleic acids such as nucleic-acid catalysts (ribozymes/deoxyribozymes)for which the method will have immediate application. In at rend similar to the establishment of longrange distance measurements in EPR, it is expected that progress in ENDOR spectroscopy will improve resolution and that mathematical procedures will be developed to extract distance distributions. |
Online CBT life skills programme for low mood and anxiety: study protocol for a pilot randomized controlled trial
Background: Low mood is a common mental health problem with significant health consequences. Studies have shown that cognitive behavioural therapy (CBT) is an effective treatment for low mood and anxiety when delivered one-to-one by an expert practitioner. However, access to this talking therapy is often limited and waiting lists can be long, although a range of low-intensity interventions that can increase access to services are available. These include guided self-help materials delivered via books, classes and online packages. This project aims to pilot a randomized controlled trial of an online CBT-based life skills course with community-based individuals experiencing low mood and anxiety.Methods: Individuals with elevated symptoms of depression will be recruited directly from the community via online and newspaper advertisements. Participants will be remotely randomized to receive either immediate access or delayed access to the Living Life to the Full guided online CBT-based life skills package, with telephone or email support provided whilst they use the online intervention. The primary end point will be at 3 months postrandomization, at which point the delayed-access group will be offered the intervention. Levels of depression, anxiety, social functioning and satisfaction will be assessed.Discussion: This pilot study will test the trial design, and ability to recruit and deliver the intervention. Drop-out rates will be assessed and the completion and acceptability of the package will be investigated. The study will also inform a sample size power calculation for a subsequent substantive randomized controlled trial.Trial registration: ISRCTN ISRCTN12890709
# Background
Depression is common, with an estimated 350 million people experiencing this mood disorder worldwide. It has a significant impact on health, wellbeing, employment status and social functioning and is the primary cause of disability globally. Various interventions are available for the treatment of depression, such as antidepressant medication and talking therapies, including low-intensity guided self-help interventions as well as high-intensity interventions, often delivered face-to-face, such as individual high-intensity cognitive behavioural therapy (CBT), interpersonal therapy and behavioural activation.
However, only between 10 % and 50 % of individuals receive appropriate treatment for their depression within health services [bib_ref] The treatment gap in mental health care, Kohn [/bib_ref]. This treatment gap may depend on a number of factors, including non-recognition of the problem (by the individual or healthcare workers), limited knowledge about treatment options and a lack of mental health resources, resulting in long waiting lists. A key aim of the WHO mental health Gap Action Programme is to increase access to psychological therapies via non-expert support or healthcare workers. This could be achieved through the application of a low-intensity CBT guided self-help approach, delivered via bibliotherapy or online in the form of computerized CBT.
Various free and licensed computerized CBT packages are available, including 'Moodgym' , 'Beating the Blues' and 'Living Life to the Full' (LLTTF). Studies show varying results regarding effectiveness and adherence to online interventions [bib_ref] Computerized cognitive behavior therapy for depression and anxiety update: a systematic review..., Kaltenthaler [/bib_ref] [bib_ref] Delivering interventions for depression by using the internet: randomised controlled trial, Christensen [/bib_ref]. A recent meta-review of the effectiveness of computerized CBT for depression found insufficient evidence to indicate which packages achieve better outcomes, indicating a need for further randomized controlled trials (RCTs) of computerized CBT to establish effectiveness [bib_ref] Meta-review of the effectiveness of computerised CBT in treating depression, Foroushani [/bib_ref].
The LLTTF course puts bibliotherapy and life skills training at the centre of the course, providing various ways of completing the course (reading, audiovisual modules, video), presenting a blended learning approach. The resources, which address key aspects of low mood and stress, take the form of classes [bib_ref] A community-based pilot randomised controlled study of life skills classes for individuals..., Mcclay [/bib_ref] [bib_ref] A community-based group-guided self-help intervention for low mood and stress: study protocol..., Mcclay [/bib_ref] McClay, Matthews, Haig, McConnachie, Morrison. A randomosied controlled trial of a community based group guided self-help intervention for low mood and stress. Submitted], printed books [bib_ref] Guided self help cognitive behavioural therapy for depression in primary care: a..., Williams [/bib_ref] and e-books, in addition to the online course. Importantly, the online version of the LLTTF intervention adheres to the key NICE recommendations regarding computerized CBT packages in terms of content, number of modules and the support element provided. A previous non-randomized feasibility study suggests that the online LLTTF course helps improve mood and anxiety; this study compared LLTTF, Beating the Blues and a book-based bibliotherapy intervention, demonstrating equivalent significant improvements across the three interventions [bib_ref] Comparative, clinical feasibility study of three tools for delivery of cognitive behavioural..., Pittaway [/bib_ref]. However, further research is needed regarding the efficacy of LLTTF in its online format as to date there have been no adequately powered RCTs of the LLTTF package. Also, following earlier evaluations of LLTTF, the intervention has been refined in three key ways: (1) content has been updated; (2) the delivery platform (website) has been enhanced to add planning and reminder components, alongside the option to read the online linked books and (3) automated support emails as well as a structured support algorithm have been developed. The extent and timing of support is known to improve outcomes in online treatments for depression, with maximal response (a Cohen's d of 0.76) when support is offered both before and during treatment, compared with 0.21 when there is no therapist support before or during treatment, 0.44 when there is therapist contact before treatment, and 0.58 when there is therapist contact during treatment only [bib_ref] Internet-based psychological treatments for depression, Johansson [/bib_ref]. We have used the conservative estimate of effect of 0.58 to provide our sample size estimate.
## Aims
In this pilot study, the primary aim will be to investigate take-up, drop-out and completion rates of the online course and the completion rates for data collection. Secondary outcomes will be mood ratings at 3 months. We will use changes in the Patient Health Questionnaire 9 [bib_ref] Validation and utility of a self-report version of the prime-MD: the PHQ..., Spitzer [/bib_ref] score to provide data relating to the effect of the intervention on depression levels. This will give an indication of efficacy, together with the drop-out and retention rates.
## Research questions primary question
Is the study design feasible? Is it possible to recruit from the community, remotely randomize participants, deliver the online intervention with telephone or email support and collect data at baseline and 3 months after randomization?
## Secondary questions
1. To what extent will participants demonstrate fidelity to the online intervention? 2. Is the LLTTF online package acceptable to participants? 3. Can we confirm our ability to recruit, and achieve the required sample size in the future substantive study?
# Methods
## Overview
The study will be a parallel, two-arm pilot RCT with a 50:50 allocation ratio across the two groups. Participants will be remotely randomized to receive the online intervention immediately (immediate-access group) or after a 3 month delay following randomization (delayed-access group). The delayed-access group will serve as the control at the primary outcome point.
## Participants
## Procedure for recruitment
People with clinical levels of depressive or anxiety symptoms will be recruited via a number of communitybased methods (newspaper advertisements in the Metro and local papers, advertisement through the Action on Depression charity, including their websites, a phone support line, newsletters and local groups), supplemented by posters, and options such as Google advertisements. No participants will be recruited directly via the NHS and we will recruit exclusively from the UK. Informed consent will be collected from all potential participants prior to entry into the study.
## Inclusion criteria
Aged 18 or over. Living in the UK. Ability to understand written and spoken English language. Regular access to a computer, smart phone or tablet with audio and broadband connection. Score of 10 or more on the Patient Health Questionnaire 9.
## Exclusion criteria
High rating of suicidality (i.e., scoring 2 or 3 on item 9 of the Patient Health Questionnaire 9). Currently receiving any psychological intervention, such as counselling or psychotherapy. New or altered dose of antidepressant in the past month.
Taking part in any other research projects.
## Sample size
We aim to recruit up to 50 participants, consistent with the widely accepted sample size of 30-50 participants for pilot studies [bib_ref] Optimal sample sizes for Phase II clinical trials and pilot studies, Stallard [/bib_ref]. We have taken an estimated effect size of 0.58 to represent a typical response rate to supported or guided online interventions, such as that tested in the proposed study [bib_ref] Internet-based psychological treatments for depression, Johansson [/bib_ref]. With a power of 0.58 and a significance level of 0.05, 48 patients per group (total n = 96) would be required. Therefore, recruiting 30-50 patients in this pilot trial should be sufficient to enable us to determine whether we can recruit and randomize sufficiently, as well as gauging our ability to retain participants and record follow-up data. Drop-out rates in internet studies can vary from 1 % to 50 %. We are also planning to record suggested predictors (severity, chronicity and treatment length) [bib_ref] Adherence in internet interventions for anxiety and depression, Christensen [/bib_ref].
## Randomization
Participants will complete initial questionnaires, and those that are eligible and have consented will then be remotely randomized to either an immediate-access or delayed-access group by the Robertson Centre for Biostatistics. Participants' ID numbers will be passed to a separate researcher who will use the randomization function in Excel to assign participants remotely to the immediate immediate-access or delayed-access group. As this is a pilot study, we will not stratify for any variables during randomization. The randomization will be done remotely by a researcher who will not be involved in the final data analysis. After randomization, the two groups of participants will be followed up in exactly the same way. Those in the immediate-access group will be given an access code and link to the LLTTF website, including instructions on using the resources. Those in the delayed-access group will be told that they will be contacted again in 3 months; the access code will be sent once follow-up data have been received.
## Intervention
The free-access online LLTTF course consists of eight modules that teach a range of CBT-based life skills.
The eight sessions are as follows:
Session 1: 'Why do I feel so bad?' -An introduction to the CBT model and the 'vicious cycle' of low mood that aims to help participants understand why they feel the way they do and how their thoughts, feelings, physical symptoms and behaviour are linked. Session 2: 'I can't be bothered doing anything' -This module focuses on the impact of reduced activity.
Users are encouraged to consider the things that they have stopped or reduced doing as a result of their low mood and make a plan to re-establish these activities in order to improve their mood. Session 3: 'Why does everything always go wrong?' teaches skills to help tackle negative thinking, including how to label unhelpful thoughts, identify negative thinking patterns and to turn these thoughts around to create more helpful ones. Session 4: 'I'm not good enough' -This session teaches how self-confidence is developed and teaches confidence-building techniques. Session 5: 'How to fix almost everything' -A problemsolving approach using an 'easy four-step plan' to help break down problems into smaller parts in order to overcome challenges. Session 6: 'The things you do that mess you up' -This session addresses unhelpful behaviours that may be worsening mood. Participants are encouraged to recognize problem behaviours such as isolating themselves, or drinking or smoking too much, and then create a plan for reducing them. Session 7: ' Are you strong enough to keep your temper?' -Participants learn to recognize the things that cause them to feel irritable or angry and the early warning signs they experience when they start to feel angry. They then learn techniques for better managing their anger to help them react differently to challenging people and situations. Session 8: '10 things you can do to feel happier straight away' -The final module teaches key lifestyle choices that can improve mood, including healthy eating, exercise and closeness with others.
Additional course worksheets, video and audio (relaxation) resources supplement the main modules. These are available on the intervention website.
## Nice specifications
The NICE guidelinesfor the treatment of adults with depression advise on the content of online selfhelp packages. These guidelines state that such interventions should:
1. Contain age-appropriate written resources -The LLTTF materials use everyday language and avoid technical CBT terms. Worksheets are colourful and often include illustrations, and can be downloaded free of charge from the website. The linked module books can be either purchased from bookshops or online, or borrowed from many libraries. 2. Contain six to eight sessions to be completed over 9-12 weeks -The LLTTF package contains eight sessions, of which the first four sessions are considered the 'core' content (formulation, behavioural activation, thinking and confidence, which brings together thinking and behaviour changes).
## Have tasks to be completed between sessions
(homework) -A planner and review sheet is used in each module to encourage participants to plan the skills that they will practise or the advice that they will follow in the coming week. 4. Include thought and behaviour change and monitoring -The LLTTF package addresses helpful and unhelpful behaviour, increasing activity and a module focused on monitoring and changing unhelpful thinking patterns. 5. Have a support element, including monitoring and reviewing progress -Support in the package includes weekly reminders (automated emails) plus support sessions with a support worker delivered weekly via email or telephone. The amount of support time allocated can be varied to reflect the complexity of the needs of the person being supported [bib_ref] Impact of support on the effectiveness of written cognitive behavioural self-help: a..., Farrand [/bib_ref]. The support is protocol drivenand focuses on encouraging use and application of the intervention. It is designed to encourage an individualized plan to be made at the end of each session using a 'plan, do, review' structure (worksheets are completed), with a focus on participants applying the skills and techniques that they have learned and making changes in their lives; using the materials to support them in doing this. To assess fidelity to the support model in the study, copies of all email supports and recordings of any phone support contacts will be made (with consent) to enable a sample of 20 (5 %) of the support sessions to be evaluated using a previously used adherence and competency checklist.
## Risk management
A potential risk to participants is that they might experience an increase in their feelings of low mood or anxiety during the study. Such risks will be addressed in several ways:
1. Active suicidal risk is included in the exclusion criteria at the screening stage. Once recruited, any participant who is found to have active risk will be signposted to support through a general practitioner, the NHS 111 freephone service or the Samaritans helpline. 2. The study website includes details of sources of urgent help. 3. The trained support workers (staff or volunteers from the Action on Depression charity) are experienced in working with such depressed or anxious people and will be able to assist any individuals who may need additional help during the study by providing information regarding how to access alternative forms of support, and the appropriate action to be taken if the participants feel they need extra help.
## Follow-up data collection
The primary outcome point will be at 3 months after randomization. Given that the focus of this pilot study is on assessing the feasibility of the trial design, no additional follow-up points will be examined. However, depending on the results of this pilot study, progression to a future Phase III definitive RCT will include follow-up to 12 months. This research plan follows Medical Research Council guidance for evaluating complex interventions. In the future substantive study, we will also plan to collect data at 6 months, and possibly one year, to record the sustainability of any improvements. Since the current study is a small pilot study, we are not able to test data collection at these longer time points. Baseline measures include age, sex, ethnicity, mood, social function, and past and current sources of support. After 3 months, all randomized participants will be invited to repeat mood and social functioning questionnaires. Use of the intervention (using site login statistics) and satisfaction in the immediate-access group will be assessed and the collection of data for an economic analysis will be piloted (these data will not be analyzed). We will also assess contamination regarding other support received and whether anyone in the delayed-access group has accessed the LLTTF course or other similar resources, or otherwise had access to CBT.
## Outcome measures
The key outcomes of the study will be the success of recruitment, retention (a minimum of 75 % follow-up rate), acceptability (as measured by drop-out rates and a standardized questionnaire) and ability to gather data.
The measures used to assess the impact on mood and social functioning, as well as satisfaction with the intervention, are described next.
## Patient health questionnaire 9
This is an open-access mood rating questionnaire consisting of nine questions mirroring DSM-IV depression diagnostic criteria and each rated from 0 to 3, giving a maximum score of 27 [bib_ref] Validation and utility of a self-report version of the prime-MD: the PHQ..., Spitzer [/bib_ref]. Cut-off scores are used to label depression severity as: 0-4, minimal depression 5-9, mild depression 10-14, moderate depression 15-19, moderately severe depression 20-27, severe depression
## Generalized anxiety disorder 7
This is a seven-item questionnaire focusing on symptoms of anxiety [bib_ref] A brief measure for assessing generalized anxiety disorder: the GAD-7, Spitzer [/bib_ref]. Each item is rated according to the frequency of the described problem in the past 2 weeks. The responses are scored as follows: 0 = 'not at all' , 1 = 'several days' , 2 = 'more than half the days' , 3 = 'nearly every day'. There is a maximum score of 21. Scores of 0-5 on this measure indicate mild anxiety; 6-10, moderate anxiety; 11-15, moderately severe anxiety and 15-21, severe anxiety.
## Work and social adjustment scale
The Work and Social Adjustment Scale [bib_ref] The Work and Social Adjustment Scale: a simple measure of impairment in..., Mundt [/bib_ref] assesses social functioning. It is a five-point questionnaire and addresses issues relating to an individual's everyday life and functioning and the impact of the individual's mood disorder on these areas. Responses are given on a scale of 0 to 8, with higher scores indicating a higher level of impairment. Those scoring over 20 are likely to have significant problems in their social functioning.
## The client satisfaction questionnaire
This is an eight-item questionnaire, rated using a four-point Likert scale [bib_ref] Assessment of patient satisfaction: development and refinement of a service evaluation questionnaire, Nguyen [/bib_ref]. It will be administered after the intervention to the immediate-access group only as a measure of satisfaction. Scores range from 8 to 32, with higher scores indicating greater satisfaction with the intervention in question. The internal consistency of the questionnaire, measured by Cronbach's α coefficient, ranges from 0.83 to 0.93 [bib_ref] Assessment of patient satisfaction: development and refinement of a service evaluation questionnaire, Nguyen [/bib_ref].
# Statistical analysis
Recruitment, uptake and adherence to intervention will be summarized and presented as percentages. Baseline characteristics will be summarized for all participants as well as for the immediate-access and delayed-access groups separately. Two-sample t tests or Mann-Whitney tests, depending on distributions, and Fisher's exact tests will be used to compare the two treatment groups and confirm that participants had been effectively randomized.
To assess efficacy, appropriate linear models will be used, using an intention-to-treat approach. Models will be adjusted for age, sex and antidepressant use at baseline. These models will be used to estimate and test the statistical significance of the within-group change between baseline and 3 months, and the between-group differences at each time point. Rates and characteristics of drop-out will be explored. Progression criteria to a full RCT would be based on adequate recruitment, evidence of possible clinical efficacy and ability to deliver the intervention and collect research data.
# Discussion
## Importance of the project
Depression is the main contributor to the global burden of diseaseand has a significant impact on sufferers' lives. Despite the fact that there are various evidencebased interventions for depression, fewer than 50 % of those affected receive appropriate treatment for low mood [bib_ref] The treatment gap in mental health care, Kohn [/bib_ref]. Low-intensity interventions, such as online CBT courses can potentially bridge the gap between onset of depression and receiving appropriate treatment by increasing access to psychological therapies. The same LLTTF content has already been evaluated in its course (group classes) and book forms, showing significant improvements in depression, anxiety, quality of life and high levels of satisfaction [bib_ref] A community-based pilot randomised controlled study of life skills classes for individuals..., Mcclay [/bib_ref] [bib_ref] Guided self help cognitive behavioural therapy for depression in primary care: a..., Williams [/bib_ref] McClay, Matthews, Haig, McConnachie, Morrison. A randomised controlled trial of a community based group guided self-help intervention for low mood and stress. Submitted]. The current pilot study will, however, represent the first RCT of the updated LLTTF online package and provide key information across a range of methodological uncertainties required for a full RCT of the LLTTF online package. These include community recruitment, uptake, delivery, adherence to the intervention and data collection, with results being used to determine the sample size needed for a substantive RCT.
## Benefits to participants
Participation within this research will allow adults who are experiencing mild to moderate symptoms of low mood or anxiety to use online CBT-based life skills and other educational modules aimed at addressing these issues. Previous research on other variants of the same course suggests that the LLTTF approach can be effective in aiding a variety of problems related to low mood and stress [bib_ref] A community-based pilot randomised controlled study of life skills classes for individuals..., Mcclay [/bib_ref] [bib_ref] Guided self help cognitive behavioural therapy for depression in primary care: a..., Williams [/bib_ref] McClay, Matthews, Haig, McConnachie, Morrison. A randomised controlled trial of a community based group guided self-help intervention for low mood and stress. Submitted]. The satisfaction data will help us to gain a key insight into how online websites can be best delivered and supported in the community.
## Working with the voluntary sector
In this pilot study, the Scottish charity Action on Depression will deliver the support element of the intervention using a pre-established support protocol. Support workers from this depression charity are experienced in working with the proposed population and have experience of introducing and supporting self-help materials. They can also assist any individuals who may need further help during the study by signposting them to additional support if there is concern about a participant's wellbeing. Action of Depression have an effective system for identifying and supporting individuals in the community and the involvement of this charity may aid in accessing hard-toreach individuals, including young men and minority ethnic groups; as well as individuals who may prefer voluntary sector support rather than traditional treatment options offered via the NHS.
## Dissemination
We aim to disseminate the findings of this pilot study in an open-access journal and via conference presentations. The journal publication will present the study findings, with the data being used to inform a future RCT protocol and a grant application. A short summary of the findings of the study will be posted online via social media accounts. We will also disseminate findings via newsletters at www.livinglifetothefull.com, which receives around 30 million hits a year from members of the public and practitioners and our partners Action on Depression.
## Trial status
The website has been designed and tested. Recruitment is expected to begin in September 2015, with the collection of outcome data starting in December 2015.
AcknowledgementsWe would like to acknowledge the assistance of the Action on Depression charity for their assistance with the study design, recruitment and support delivery.FundingThe project is being sponsored by the University of Glasgow, funded by NHS Greater Glasgow and Clyde and is being carried out within the Institute of Health and Wellbeing.AbbreviationsCBT: cognitive behavioural therapy; LLTTF: 'Living Life to the Full'; NICE: National Institute for Health and Care Excellence; RCT: randomized controlled trial; WHO: World Health Organization.Competing interests CW is an author of a variety of written and computerized CBT resources. These are licensed through Five Areas Ltd, a company that delivers free and licensed online life skills resources based on a CBT model in a variety of settings, including the NHS and voluntary and private sectors. The other authors declare that they have no competing interests.Authors' contributions CW is the chief investigator of the study, contributed to the writing of the study protocol, is the clinical lead for the trial, will supervise the support workers and will contribute to the interpretation and reporting of the findings. C-AMcC contributed to the writing of the study protocol and will carry out recruitment, screening and randomization of participants, liaise with the Action on Depression support worker, facilitate access to the intervention and collect and interpret follow-up data. CH is the statistician for the study, carried out the power calculation for the project, assisted with study design decisions and will provide statistical support in the analysis of the final outcome data. PF contributed to the writing of the study protocol, will share his trial expertise during the study and will contribute to the interpretation and reporting of the findings. RJ assisted with the design of the study, will share his trial expertise during the study and will contribute to the interpretation and writing up of the findings. JM contributed to the conceptualisation of the study, will share her depression research and RCT expertise during the study and will contribute to the interpretation and writing up of the findings. RM contributed to the development of the research design and will use her clinical and research experience to assist with the trial and the interpretation of the findings. All authors are members of the research team, were involved in the preparation of this manuscript and will interpret and report the final results. All authors read and approved the final manuscript.Authors' informationProfessor Christopher Williams, Professor of Psychosocial Psychiatry, University of Glasgow has experience and skills in using and evaluating self-help materials and running large randomized controlled studies, in primary care and the community. Carrie-Anne McClay is a PhD student with experience of carrying out community-based RCTs, specifically in depression and eating disorders. Professor Paul Farrand, Director of Postgraduate Training in LI CBT, University of Exeter has experience and skills in developing and evaluating self-help materials and running pilot and feasibility randomized controlled studies, in primary care, community and IAPT settings. Professor Jill Morrison, Professor of General Practice is an academic general practitioner with an interest in primary care management of depression and is experienced in running RCTs in community-based settings. Dr Caroline Haig is a biostatistician at the Robertson Centre for Biostatistics, University of Glasgow and is experienced in the design, analysis and reporting of randomized studies. Ray Jones, Professor of Health Informatics, has experience of novel online recruitment methods and RCTs of online interventions. Rebeca Martinez is a consultant psychiatrist at Merseycare NHS Trust, with experience of the management of depression in clinical practice.- We accept pre-submission inquiries - Our selector tool helps you to find the most relevant journal Submit your next manuscript to BioMed Central and we will help you at every step: |
Development of an education and self-management intervention for chronic headache – CHESS trial (Chronic Headache Education and Self-management Study)
Background: Self-management interventions are well recognised and widely used in chronic conditions. Their application to chronic headaches has been limited and generally of low quality. We describe here our process for developing an evidence based, and theory driven, education and self-management intervention for those living with chronic headache. Methods: Our intervention was designed using several core information sources; the results of three systematic reviews, qualitative material from those living with chronic headaches, our knowledge from existing self-management interventions; and finally collaborative input from a multidisciplinary team of clinicians, academics, patients, and charity partners. We manualised the intervention and associated training as a package for use in a feasibility study. We made adaptations for its use in a randomised controlled trial.Results:We piloted the intervention in four groups with a total of 18 participants. Qualitative feedback from 12 participants and five facilitators allowed the intervention to be refined for the main randomised controlled trial. Some of the key changes included shortening of the overall intervention, changes to the originally planned facilitators and spreading the facilitator training over three days rather than two. We are now testing the final revised intervention in a randomised controlled trial of its clinical and cost effectiveness. The group component of the intervention is delivered over two days with the first day focused on living, understanding and dealing with chronic headaches and the second day exploring how to adapt and take control of one's life with chronic headaches.Conclusion: Our pilot work indicates that our intervention is feasible to deliver, and with the relevant changes would be acceptable for use with this population. Our randomised control trial is ongoing. We anticipate publishing final results in 2021.Trial registration: ISRCTN79708100. Registered 16th December 2015, http://www.isrctn.com/ISRCTN79708100
# Background
Chronic headaches affect 2-3% of the population. Globally migraine, medication overuse headache and tension type headache are, respectively, the second, sixth and 12th leading causes of disability from neurological disorders. Around 4% of UK primary care consultations and 30% of neurology outpatient appointments are due to headache disorders [bib_ref] Primary headache disorder in the emergency department: perspective from a general neurology..., Gahir [/bib_ref] [bib_ref] A record of patient encounters in neurological practice in the United Kingdom, Hopkins [/bib_ref] [bib_ref] Headache and migraine in primary care: consultation, prescription, and referral rates in..., Latinovic [/bib_ref]. The annual direct treatment cost to the UK National Health Service (NHS) is £1 billion [bib_ref] How do patients referred to neurologists for headache differ from those managed..., Ridsdale [/bib_ref]. As headache disorders are most prevalent among the working age population they have a large economic impact. The annual cost of headache disorders in the UK is between £5-7 billion; primarily due to lost production.
Managing chronic headaches can be challenging, the focus has tended to be on pharmacological interventions. Pharmacological management has tended to focus on episodic migraine, with few studies focusing on chronic headaches [bib_ref] A comparative effectiveness meta-analysis of drugs for the prophylaxis of migraine headache, Jackson [/bib_ref]. Only topiramate and Onabotuli-numtoxinA have been shown to reduced headache days for those with chronic migraine [bib_ref] OnabotulinumtoxinA for treatment of chronic migraine: pooled results from the double-blind, randomized,..., Dodick [/bib_ref] [bib_ref] Efficacy and safety of topiramate for the treatment of chronic migraine: a..., Silberstein [/bib_ref] [bib_ref] Topiramate reduces headache days in chronic migraine: a randomized, double-blind, placebo-controlled study, Diener [/bib_ref] [bib_ref] Topiramate and triptans revert chronic migraine with medication overuse to episodic migraine, Mei [/bib_ref] [bib_ref] Topiramate in the treatment of chronic migraine, Silvestrini [/bib_ref]. The impact on quality of life has not been explored in trials and the overall use of other pharmacological strategies is very limited and of poor quality amongst a chronic headache population. The UK National Institute for Health and Care Excellence (NICE) published guidance on headaches in September 2012. Apart from a recommendation to consider a course of acupuncture for people with chronic migraine or chronic tension type headache, the guideline developers did not find suitable evidence to allow recommendations on non-pharmacological treatments for people with chronic headache .
There is good evidence for supportive self-management programmes for long-term conditions. Such programmes have been used in a range of chronic conditions [bib_ref] Group based training for self-management strategies in people with type 2 diabetes..., Deakin [/bib_ref] [bib_ref] Systematic meta-review of supported self-management for asthma: a healthcare perspective, Pinnock [/bib_ref] [bib_ref] Self management for patients with chronic obstructive pulmonary disease, Zwerink [/bib_ref]. These use patient education and behaviour change strategies to encourage those living with chronic conditions to engage and take an active role in managing their own condition and to minimise the impact this condition has on individual's physical and psychological functioning. Chronic conditions can have a substantial impact on individual's lives [bib_ref] Quality of life in migraine patients: a qualitative study, Ruiz De Velasco [/bib_ref] [bib_ref] Perceptions and needs of patients with migraine: a focus group study, Cottrell [/bib_ref] therefore a focus on a biopsychosocial approach taking into account physical, psychological and social factors is appropriate [bib_ref] A review of chronic pain impact on patients, their social environment and..., Duenas [/bib_ref]. There has been limited high quality evidence for the use of self-management interventions in the treatment of chronic headaches [bib_ref] Non-pharmacological self-management for people living with migraine or tension-type headache: a systematic..., Probyn [/bib_ref] , hence the need for further research.
There is some suggestion of an association between chronic headaches and chronic musculoskeletal pain [bib_ref] Prevalence of neck and low back pain in community-dwelling adults in Spain:..., Fernandez-De-Las-Penas [/bib_ref] [bib_ref] Self-reported comorbid pains in severe headaches or migraines in a US national..., Plesh [/bib_ref]. A systematic review of the association between headache and low back pain found that the odds ratio for the association ranged from 1.55 to 8.0 in different studies. This association maybe linked to central sensitisation, which may provide a common pathway for chronic headache and other chronic pain syndromes [bib_ref] Mechanism of chronic migraine, Aurora [/bib_ref] [bib_ref] Gray matter changes related to chronic posttraumatic headache, Obermann [/bib_ref]. It is therefore appropriate to draw on the current evidence base from other painful chronic conditions to inform strategies to facilitate effective self-management of chronic headaches.
Building on this evidence we have been funded to develop and test a group self-management support programme for people living with chronic headaches (funded by the NIHR Programme Grants for Applied Research programme -RP-PG-1212-20,018) [bib_ref] Usual care and a self-management support programme vs usual care and a..., Patel [/bib_ref]. We describe here the development and initial evaluation of an education and self-management support intervention for people living with chronic headache. The specific aim of the programme is to enable people with chronic headache to manage their pain better and to improve their quality of life. For our main randomised controlled trial, we are testing the hypothesis that amongst adults with chronic headache, the provision of a self-management support programme in addition to best usual NHS care will help to improve headache related quality of life.
# Methods
The Medical Research Council framework for designing complex interventions, the Person-Based Approach and core theoretical principles from psychological models including Michie's behaviour change wheel and taxonomy have guided the development of our intervention [bib_ref] The behavior change technique taxonomy (v1) of 93 hierarchically clustered techniques: building..., Michie [/bib_ref] [bib_ref] The behaviour change wheel: a new method for characterising and designing behaviour..., Michie [/bib_ref] [bib_ref] Developing and evaluating complex interventions: the new Medical Research Council guidance, Craig [/bib_ref]. The MRC framework encourages the development of interventions by drawing on theory and evidence based research. The Person-Based Approach suggests behaviour change intervention should be grounded in a detailed insight of the needs, perspectives and context of individuals who will be the recipients. We have adapted the Person-Based Approach and used it as a structure here. [fig_ref] Table 1: An overview of our intervention development process using an adapted version of... [/fig_ref] provides an overview of our intervention development process.
Throughout the process of designing this intervention, we have drawn upon the views and opinions of our PPI partners to make sure what is produced would be acceptable and useful for those living with chronic headaches. Our PPI support comes from key members of the leading headache charities, members attending our intervention design day, our lay facilitators (who themselves live with chronic headaches) and a wider PPI reference group who have volunteered to support the overall CHESS study with PPI needs.
The development and initial evaluation of the intervention package was just one component of the CHESS feasibility study. A detailed account of the full feasibility phase are described elsewhere.
## Planning phase -systematic reviews
We completed three systematic reviews to help inform the development of the intervention. Full details of the methodology and results of these reviews are reported elsewhere [bib_ref] Non-pharmacological self-management for people living with migraine or tension-type headache: a systematic..., Probyn [/bib_ref] [bib_ref] The lived experience of chronic headache: a systematic review and synthesis of..., Nichols [/bib_ref] [bib_ref] Prognostic factors for chronic headache: a systematic review, Probyn [/bib_ref]. Here we have provided a summary of the key finding and the influence these had on our intervention design [fig_ref] Table 2: Summary of results from reviews and influences on intervention design [/fig_ref].
## Planning phase -qualitative interview study
We conducted qualitative interviews to inform the intervention design. This interview data and the lived experience systematic review both aimed to ensure that the intervention design included a strong patient focus. Migraine Action, 1 one of the charity partners in CHESS, sent letters on the trial's behalf to their members within a predefined geographical region for ease of travel (100 members resided in this area). Interviews were face to face in people's homes and were audio recorded after taking informed consent. Topic guides explored their experiences of what might be helpful, or unhelpful, treatment strategies and where they sought information regarding their headaches. Interviews were transcribed, anonymised and analysed thematically. All data were collected and held in accordance with data protection guidelines.
From the 100 invitation letters sent out, we received 21 responses. Of these responses, five had headaches for < 15 days per month, three had no headaches, three were under the age of 18 years and two were not interested in the study. Of the eight that met our inclusion criteria of headaches on > 15 days per month for at least three months, one had new daily persistent headache and was therefore excluded. We interviewed seven people; the results of these interviews were presented at the intervention development day.
When participants were asked about things that they had tried which were helpful for their headaches they spoke about a vast array of treatments and strategies. These included; belonging to organisations for information and support, seeing approachable and knowledgeable doctors, seeing different therapists (physiotherapists, acupuncturist, counsellor, craniosacral therapist), meditation/relaxation, distraction techniques, being outdoors, having social support from people who understand, having a positive mind-set, Yoga, Pilates and breathing techniques. However, what was helpful to some was often unhelpful to others and many of the things tried were out of the scope of our intervention.
When describing their medication use, interviewees spoke of 'mixing and matching' , and 'trial and error' , some medication working at one time and not at another. Side effects were an important feature and two interviewees voiced worries about their medication being rationed. Exact medication use was difficult to ascertain at interview. This was an area for us to explore further in the feasibility study interviews.
We asked participants for their views about what we should consider for inclusion in an education and self-management group intervention. Suggestions included getting peer support (group meetings); developing skills in stress management, relaxation/meditation; Synthesis of evidence using systemic reviews: a. education and self-management interventions for chronic headache [bib_ref] Non-pharmacological self-management for people living with migraine or tension-type headache: a systematic..., Probyn [/bib_ref] b. lived experiences review [bib_ref] The lived experience of chronic headache: a systematic review and synthesis of..., Nichols [/bib_ref] c. prognostic factors for chronic headache [bib_ref] Prognostic factors for chronic headache: a systematic review, Probyn [/bib_ref] Qualitative material collected via interviews with people living with chronic headache
## Design
Outline of needs and challenges to be addressed to meet overall intervention objectives
Creating an outline of the intervention aims and objectives including the key features and components to achieve the objectives using: Style and content review [bib_ref] Non-pharmacological self-management for people living with migraine or tension-type headache: a systematic..., Probyn [/bib_ref] To review the effectiveness of self-management interventions for headaches and highlight the differential components included and delivery methods used
Inclusion of CBT The overall intervention is informed by the core principles of cognitive behavioural therapy (CBT). The focus being on unhelpful thinking patterns and the need to recognise such thought processes and look for alternatives that are more helpful. Participants have the opportunity to explore the different types of unhelpful thought patterns and subsequently reflect on the challenges these create and ways to make them more helpful/manageable.
## Inclusion of education
The programme is an educational and self-management intervention and therefore includes topics that carry a large educational component. This includes topics such as 'headache information and mechanisms' and 'medication management.'
Inclusion of mindfulness As part of a taster session, mindfulness is included. Participants are provided with a mindfulness CD for home practice.
## Inclusion of relaxation
Relaxation is included as a taster session and participants are provided with a copy of the relaxation CD for home practice.
Group interventions more effective The intervention is group based, aiming to get between 8 and 10 participants per group.
Face to face and remote delivery did not make much of a difference As this is a complex intervention with several components, we felt a face-to-face, group intervention with a built in one to one consultation would be the best option based on previous experience from the team in delivering complex interventions.
## Homework -no difference in studies offering this and not
We included homework as part of our intervention to enable bedding in of information and discussions from day one and to allow any uncertainties to be clarified on day 2. Participants are encouraged to make use of the relaxation CD and to watch the headache DVD.
Email/telephone supportno difference in studies offering this and not Telephone follow-up is provided as a means of supporting those who are implementing changes and in particular those who might be withdrawing from medication. The frequency of these calls are individually negotiated between the nurse and the participant.
No indication that delivery by a Psychologist or Psychotherapist was any more or less effective than a nurse or Allied Health Professional (AHP)
The collaborative team carefully considered who should facilitate the delivery of the intervention. Due to the medical aspects of headaches around mechanisms, medication and headache classification a nurse was deemed most appropriate.
Lived experiences review [bib_ref] The lived experience of chronic headache: a systematic review and synthesis of..., Nichols [/bib_ref] To synthesis the qualitative literature on the lived experience of people with chronic headache disorder Headaches act as a driver to increase medication We have specifically included a session on medication to allow exploration of acute and preventative medication. Focus is also given to the concept of medication overuse headaches and subsequently the opportunity to discuss this during a one to one consultation.
Headaches lead to avoidance in planning The intervention includes the headache pain cycle and the need to break the cycle. We explore the skills associated with identifying barriers to change and using problem solving and goal setting as a means to engaging in meaningful activity. Participants are encouraged to complete their own goal-setting plan and to bring that to the one to one appointment for discussion.
Headaches encouraged changes in sleep patterns Sleep management is included as a session to enable participants to understand the link between sleep and thoughts and subsequently look at what is, and is not, recommended for good sleep management.
Headaches a driver to stopping doing things The headache pain cycle is used to discuss a feeling of being trapped and therefore withdrawing. This is further explored to identify strategies to help break this cycle.
A sense of loss of control The whole intervention is designed to educate and encourage those with chronic headaches to explore strategies to help them better manage their headaches and improve their quality of life. As part of this journey we explore the concept of control and the implication this can have on headaches.
learning about triggers, lifestyle factors and medication; having information in one place; having education for others such as family and employers; gaining expert support and finding out about the latest research and advances.
They also gave us some practical points about running a group for people with frequent headache, this included running of the course in a neutral environment so that it was not too medical (such as a hospital setting). They felt small groups were important and there should be regular breaks throughout the day. An option to have groups in the evening was also suggested for those that work or cannot attend during the day.
## Design phase -knowledge from existing intervention
With the associated links between chronic headaches and those with chronic pain, we have drawn upon the knowledge and experience of intervention design and delivery from those involved in the design and running of the COPERS study. Key members of that study are part of the CHESS study team. This was a randomised control trial of a group education and self-management intervention for those living with chronic pain. This was a complex intervention designed using underpinning psychological theory and the guidance of the MRC framework for designing complex interventions [bib_ref] Developing and evaluating complex interventions: the new Medical Research Council guidance, Craig [/bib_ref]. This team conducted two reviews to help inform their intervention design and the results of these reviews have been examined as part of the CHESS intervention development [bib_ref] Effective delivery styles and content for self-management interventions for chronic musculoskeletal pain:..., Carnes [/bib_ref] [bib_ref] Can we identify how programmes aimed at promoting self-management in musculoskeletal pain..., Miles [/bib_ref]. The COPERS intervention was proven to be acceptable and effective in the medium-term for depression, anxiety, social integration and support, pain acceptance, and self-efficacy in pain management. There are also long-term positive effects for depression, and social integration and support.
The basic structure and content of the COPERS intervention was used to inform the CHESS intervention as was the use of groups so that people could learn from each other and the use of lay facilitators to jointly facilitate with the nurse. Having two facilitators allows for easier management of challenges or difficulties without the need to disrupt the rest of the group. Other experience used to inform the CHESS intervention was holding courses in familiar accessible locations and that clinicians from different disciplines were capable of delivering the intervention.
## Design phase -theoretical underpinnings
We have drawn upon the core theoretical principles from several psychological theories including, many of which have been used in other self-management interventions; cognitive behavioural theory, social cognitive theory [bib_ref] Self-efficacy: toward a unifying theory of behavioral change, Bandura [/bib_ref] , acceptance and commitment therapy, theory of planned behaviour and reasoned action [bib_ref] The theory of planned behavior, Ajzen [/bib_ref] and the health belief model [bib_ref] Why people use health services, Rosenstock [/bib_ref] [bib_ref] Social learning theory and the health belief model, Rosenstock [/bib_ref]. to guide the development of the intervention aimed at our specific population. The behaviour change wheel and taxonomy [bib_ref] The behavior change technique taxonomy (v1) of 93 hierarchically clustered techniques: building..., Michie [/bib_ref] [bib_ref] The behaviour change wheel: a new method for characterising and designing behaviour..., Michie [/bib_ref] have been used to guide our thinking about the sources of behaviour that could be targeted, the rational for each topic area and the most effective strategies for implementing and encourage behaviour change. [fig_ref] Table 3: Theoretical underpinnings and behaviour change rational and techniques for CHESS intervention [/fig_ref] summarises the theoretical underpinnings and the behaviour change techniques that have [fig_ref] Table 2: Summary of results from reviews and influences on intervention design [/fig_ref] Summary of results from reviews and influences on intervention design (Continued)
## Key findings influences on intervention design
Prognostic review [bib_ref] Prognostic factors for chronic headache: a systematic review, Probyn [/bib_ref] To identify predictors of prognosis in studies of those with chronic headache Depression and anxiety The intervention includes topics around the link between mood and headaches and the impact this can have. We provide participants with a handout outlining the possible symptoms of depression and advise to seek support from their GP if they are struggling with these. Mindfulness and relaxation are built in as strategies to help manage mood and anxiety.
Medication overuse This is covered as a topic in the facilitated group sessions and then further discussed during the one to one consultation, if relevant.
## Poor sleep
The concept of a balanced and healthy lifestyle is facilitated as a topic during the group sessions. As part of this, sleep and effective sleep management strategies are discussed.
High stress Sessions on managing stress and anxiety are included. Participants are encouraged to explore the impact of stress and anxiety and subsequently look at strategies for management. Relaxation and mindfulness are introduced as strategies to manage stress and participants are encouraged to practice these at home.
Headache management self-efficacy The course is designed in inform, empower and build confidence in those with headache to take control and use self-management strategies to help them manage their headaches better. driven the design and components of the CHESS intervention. Grounded in the evidence base from our review, we built in 'taster' sessions around relaxation and mindfulness. The sessions provide participant with the opportunity to participate via instruction on how to engage in relaxation and mindfulness. Participants then have the opportunity to take away material to allow for home practice with the aim to encourage regular practice and habit formation in the long-term.
We have also produced a DVD for participants to share with family and friends. The DVD is aimed to be informative, reiterating the core messages from the course as well as portraying what it is like to live with chronic headaches. This was developed in response to suggestions from PPI members at the intervention design day as well as the qualitative review and interviews that suggested those living with chronic headaches feel others do not understand.
## Design phase -collaborative intervention design meeting
This meeting was attended by clinicians including a neurologist and two general practitioners, directors from two leading headache charities (Migraine Action; National Migraine Centre), three lay people living with chronic headaches, psychologists with expertise in self-management and behaviour change, academics and researchers (18 attendees in total). The multidisciplinary team bring together clinical expertise in the management of chronic conditions including headaches and pain as well as expertise in behaviour change and self-management. We drew on this experience to inform the development of the intervention.
The day comprised of short presentations on the results from the three systematic reviews, main findings from the qualitative interviews, an overall study summary from COPERSand a summary of the main outcomes from a classification day which took place to inform the design of a logic model to allow chronic headaches to be classified. The classification day comprised of facilitated discussions on core questions to help inform the development of a logic model. The outcomes from these small group discussions were discussed in a large plenary session and this information was used to help develop and refine a logic model to be used in the study and intervention process. Full details have been published elsewhere [bib_ref] Development and validation of a telephone classification interview for common chronic headache..., Potter [/bib_ref].
During the intervention design meeting, presentations followed facilitated discussion about the results, what the tailored education self-management intervention should look like, what ongoing support participants would need and what the control intervention should be. We have had input from the three leading headache charities. We have also had the input from three lay members who attended the intervention design day and were involved in the discussions and decisions. After the meeting, a drafting document was circulated which provided all those that attended the opportunity to feedback. This combined input has helped shape the intervention.
# Results
Here we describe the intervention package we developed for implementation based on this work and how we implemented, evaluated and refined ready for use in our RCT.
The CHESS intervention is embedded in the biopsychosocial model, which acknowledges that long-term conditions have physical, psychological and social implication on individuals and therefore management should focus on a combination of these factors. The overall aim of the course was to encourage and enable those with chronic headache to manage and cope with their pain better, to improve their quality of life despite their headache.
## Summary
To clarify learning from the two days and introduce the structure of the one to one sessions Embedding learning
One to one session with nurse
To make a classification of headache type and discuss medication management based on the classification. To also review lifestyle factors and goal setting to enable the participant to engage in behaviour change.
biopsychosocial model Provision of pharmacological information and support as well as embedded learning. Review of goals including reflection on performance and consequence of change. Selfmonitoring of headaches and subsequent health, social and environmental consequence. Social reward and positive reinforcement
## Feasibility intervention design
We designed an education and self-management intervention delivered and facilitated by a non-headache specialist nurse and lay facilitator (someone who lives with chronic headaches). The intervention was delivered over two full days (10:00-15:00) followed by a one to one consultation with the nurse facilitator (agreed at a time convenient for the nurse and participant, approximately a week after the group sessions, lasting up to two hours). This was then followed by a half-day group follow-up session (10:00-12:30). The course was designed for between 8 and 10 participants. Facilitators were recruited through adverts and local contacts. Lay people were recruited through our charity partners. We had three nurses and two lay people interested and available to attend a two day training course aimed to inform them about trial procedures, equip them with facilitation skills for running groups and familiarise them with the content of the intervention. An assessment of learning form was completed by the facilitators following the training to check their understanding, knowledge and confidence in delivering the intervention. [fig_ref] Figure 1: Course structure in the feasibility interventionFig [/fig_ref] shows the structure of the course.
The intervention was piloted in four groups in Warwickshire, England with a total of 18 participants. Three of these groups were delivered based on the course structure presented in [fig_ref] Figure 1: Course structure in the feasibility interventionFig [/fig_ref] , the fourth group was delivered using a two day structure based on the feedback that had been received. The groups were run in community settings.
We interviewed the five facilitators and 12 of the participants to capture their feedback and experience. We also collected written participant feedback at the end of the course and asked facilitators to provide written reflective logs after each day of facilitation.
Thematic analysis was used to identify common themes across the different components of the intervention. Overall, the results suggested the groups were liked and the material was deemed useful and interesting. Some people had gained new information which was personally useful and others had tried some of the self-management strategies discussed in the group. The opportunity to meet and participate in a group was much appreciated. The feedback from these interviews have enabled the intervention team to streamline the course. [fig_ref] Table 4: Summary of the main changes to the intervention following facilitator and participant... [/fig_ref] describes the main changes made following the feedback.
Some other more practical issues identified by participants included the need to carefully consider venues for delivering CHESS especially in relation to seating, temperature and lighting. Each venue selected for the CHESS delivery is assessed for suitability by the trial team. The facilitator manual provides a reminder for to facilitators to assess the temperature and lighting and to accommodate needs where possible.
In light of the feedback the structure of the intervention was revised, shows the structure for the RCT and [fig_ref] Table 5: Final modules and course content for the CHESS intervention Day Modules Content... [/fig_ref] shows the final modules and content of the intervention package.
# Discussion
The outcomes from our development and pilot work suggest the two-day group intervention followed by a one to one consultation with the nurse and relevant telephone follow-up is feasible and acceptable to a chronic headache population after the recommended changes were applied. Our training package (after the addition of a third day) is sufficient to support facilitators in developing the relevant knowledge base and confidence in facilitating groups. The development process has mapped onto the MRC framework for developing complex interventions as well as being guided by a Person-Based Approach and core theoretical principles from psychological models and behaviour change theory. We have taken into account the views and input from a range of stakeholders including clinicians, healthcare professionals, academics, patients and charity partners throughout the intervention planning, design and implementation phases.
This work has some potential weaknesses, the number of participants and facilitators was small and we might not have been able to capture the full range of Facilitators for the main randomised controlled trial During the feasibility phase it had been difficult for the lay facilitators to commit to running groups due to their own chronic headaches. This was collaboratively discussed and there was agreement to use an AHP who is registered with an appropriate regulatory body rather than a lay facilitator for the main RCT.
Length of the course The original 2.5 days was challenging for participants due to work and family commitments. For this reason the half day follow-up was removed. We felt confident that any issues could be raised during the one to one session and subsequent telephone follow-up. The content due to be covered on the follow-up day was merged into day two of the course. This was possible as other topics had been removed and/or revised. The fourth group was run using this two day format.
Timing of the sessions Originally we had planned to run the two days consecutively, with the follow-up day approximately two weeks later. Two groups were run using this format but participants struggled with some experiencing headaches. The third group was run using a weekly format with one day delivered each week with the follow-up day two weeks after the second group session. In between the second full day and follow-up session, participants had their one to one consultation with the nurse.
Merging of topics related to unhelpful thinking patterns and reframing negative thoughts to positive thoughts
Facilitators found these session difficult to facilitate as they felt disjointed. By merging the sessions it allowed more clarity on the importance of recognising unhelpful thought patterns and subsequently using questions to challenge them and look for more helpful alternatives.
Merging physical activity as a topic into lifestyle factors Physical activity was included as a separate topic, however it fitted better under the theme of lifestyle factors therefore this topic was integrated into this section.
## Removal of session on emerging and complementary treatments
The nurses felt this session was difficult to deliver because of the level of knowledge required and the questions presented to them. For this reason, as well as our understanding that most of these treatments would not be readily available on the NHS we decided to remove this session. We provided participants with a handout detailing the websites of some of the leading headache charities where they could find more information on treatments.
Addition of summary and reminders at the end of day 1 An overall summary table of the content covered was added to allow participants to reflect on the material covered and understand the links between the sessions. We also include some reminders to ensure participants continued to complete the headache diary, watched the DVD and practiced the relaxation where possible.
Changes to some of the session and module names This was done to improve clarity.
## Mindfulness session
This session was broadened to mindfulness and relaxation to give participants the opportunity to understand the differences between the two and have the opportunity to practice these at home.
## Chess intervention training
The training for facilitators in the feasibility phase was delivered over two full consecutive days. The training was delivered by two psychologists, a general practitioner, a neurologist and a nurse by background. From the feedback we found many of the facilitators struggled with the amount of information covered and that they would have liked sometime between the two training sessions. Taking into consideration the feedback, we revised the training for the RCT, it is now delivered over three days, with the first two days as consecutive days covering the group educational and self-management components. This is then followed by a third day, a week later, to cover the training for the one to one session and follow-up phone calls. Further one to one support was provided to each nurse ahead of their first one to one sessions to recap on the logic model and medication advice.
In addition, all facilitators are supported individually as and when required by the intervention design team. experience in our qualitative work. However, our experience was that no new themes were emerging once interviews were complete. We have not evaluated the revised package before implementing this in the main trial; although the opportunity exists to make minor adjustments following the internal pilot. It is disappointing that we were not able to include lay facilitators but our experience was, that for this study, this was simply not practical. The PPI element has been an important part of the intervention development process and a particular strength of this work. Our PPI partners have guided us to develop something that is acceptable and relevant for this clinical population. Our early work and the involvement of our PPI partners has allowed us to identify the needs of this population, explore the opportunity to map existing strategies from other interventions, identify any barriers and facilitators to change and subsequently identify appropriate behaviour change techniques to enable implementation of more desired behaviours for the long-term.
The process of developing the intervention to provide a manualised package for use in our definitive randomised controlled trial has been a lengthy process. Despite this, we have produced an intervention that is grounded in the needs of people living with chronic headaches as well as the theory and evidence base.
As part of the intervention development we have also considered quality assurance. For the main RCT the quality, accuracy and approach to delivery are observed and facilitators are provide with feedback. Observations are conducted by members of the study team who are familiar with the intervention. In addition to this, we encourage personal reflection from facilitators via email recording their thoughts and feelings about the sessions, noting things that went well and where things could have gone better. These emails help the intervention design team best support facilitators.
# Conclusion
The CHESS education and self-management intervention is currently being tested in a randomised controlled with Serco Ltd. related to return to work initiatives. He is a co-investigator on a study which receives support in kind from Orthospace Ltd. He is an editor of the NIHR journal series, and a member of the NIHR Journal Editors group for which he receives a fee. He has published multiple papers on chronic pain some of which are referenced in this paper.
[fig] Figure 1: Course structure in the feasibility interventionFig. 2 Structure of the CHESS intervention for the randomised controlled trial Patel et al. The Journal of Headache and Pain (2019) 20:28 [/fig]
[table] Table 1: An overview of our intervention development process using an adapted version of the Person-Based Approach [/table]
[table] Table 2: Summary of results from reviews and influences on intervention design [/table]
[table] Table 3: Theoretical underpinnings and behaviour change rational and techniques for CHESS intervention [/table]
[table] Table 4: Summary of the main changes to the intervention following facilitator and participant feedback [/table]
[table] Table 5: Final modules and course content for the CHESS intervention Day Modules Content of sessions 1. Living, understanding and dealing with chronic headaches 1. Introduction to the course and each other Session 1: Welcome and introductions Session 2: Course overview 2. Understanding chronic headaches and acceptance Session 3. Headache information and mechanisms Session 4. Acceptance of chronic headaches Taster activity -Relaxation and breathing 3. Mind, body and pain link Session 5. Impact of thoughts, mood and emotions on headaches Session 6. Headache cycle and breaking the cycle 4. Dealing with unhelpful thought patterns Session 7. Unhelpful thinking patterns: recognising and finding alternatives 5. Summary Session 8: Summary and reminders from day 1 2. Learning how to adapt and take control of your life with chronic headaches 1. Reflections Session 9. Reflections from Day 1 2. Back to basics Session 10. Identifying barriers to change and exploring problem solving and goal setting Session 11. Lifestyle factors and impact on headaches 3. Making headaches more manageable Session 12. Managing stress and anxiety Session 13. Managing sleep better Session 14. Mindfulness and relaxation for headaches Taster activity -Mindfulness practice 5. Treatment options Session 15. Medication management 6. Communicationexplaining your headaches to others Session 16. Relationships and communication with family, carers and friends Session 17. Communicating better with Health Professionals 7. Future management Session 18. Managing setbackswhat to do when things don't go to plan 8. Summary Session 19. Summary of course 3. One to one session with nurse Session covers: • Classification assessment with headache diary • Discussion around medication • Lifestyle factors and personalised goal setting. [/table]
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Effects of β2-Adrenergic Receptor Gene Polymorphisms on Ritodrine Therapy in Pregnant Women with Preterm Labor: Prospective Follow-Up Study
This study aimed to evaluate the effects of β2-adrenergic receptor (ADRB2) gene polymorphisms on ritodrine therapy outcomes in patients with preterm labor. Genotyping analysis of ADRB2 gene (rs1042713, rs1042714, rs1042717, rs1042718, and rs1042719) was performed on 137 patients with preterm labor. Survival analysis was conducted for the effects of SNPs on the median time to delivery as a primary outcome. The median time to delivery in the study patients was 349.3 h. Gestational age at admission and modified Bishop scores revealed significant effects on time to delivery (p < 0.001). Among studied SNPs, rs1042717 and rs1042718 showed linkage disequilibrium in this population, and their effects on time to delivery were marginally significant (p < 0.1). Patients with variant-homozygotes in the rs1042713 showed considerably shortened time to delivery compared to wild-allele carriers. The rs1042719 polymorphism significantly affected time to delivery in both univariate and multivariate analysis; the GC and CC carriers showed 64% decrease in time to delivery compared to the wild-type homozygote carriers. Based on the results, it was concluded that the gene polymorphisms of ADRB2 OPEN ACCESS could affect ritodrine therapy in patients with preterm labor. However, given the single-center and the relatively small sample size, our hypothesis requires further independent validation using multi-center and large sample size.
# Introduction
Preterm birth is defined as spontaneous labor occurring before 37 completed weeks of gestation. It is the leading cause of neonatal mortality and morbidity in newborns without congenital anomalies or chromosomal abnormalities [bib_ref] Annual summary of vital statistics, Hamilton [/bib_ref] [bib_ref] Infant mortality statistics from the 2003 period linked birth/infant death data set, Mathews [/bib_ref]. According to 2012 statistics, the rate of preterm birth ranges between 5% and 18% of babies born across the world [bib_ref] National, regional, and worldwide estimates of preterm birth rates in the year..., Blencowe [/bib_ref]. Preterm birth also has major socioeconomic implications with associated hospital stays [bib_ref] Multicentre, parallel group, randomised, single-blind study of the safety and efficacy of..., Shim [/bib_ref]. Tocolytic drugs with a variety of mechanisms are being used to relax the uterus and suppress the uterine contractions to prevent preterm birth.
Ritodrine is a β2-adrenergic receptor (ADRB2) agonist, resulting in uterine smooth muscle relaxation. Although it has been widely used in several European and Asian countries, the efficacy of ritodrine was not consistent [bib_ref] Multicentre, parallel group, randomised, single-blind study of the safety and efficacy of..., Shim [/bib_ref]. Numerous factors are believed to contribute to the speed and outcome of delivery, but several studies implicated the importance of association between genetic predisposition and preterm birth [bib_ref] The genetics of preterm birth: Using what we know to design better..., Weinberg [/bib_ref].
Human ADRB2 gene is encoded on chromosome 5, and its genetic variability has been widely characterized with several single nucleotide sequence variations [bib_ref] Polymorphisms of the β2-adrenergic receptor, Liggett [/bib_ref]. Especially, missense mutations encoding for amino acids 16 and 27 of the extracellular N-terminus of the β2-adrenergic receptor have been studied in many areas including β-blocker therapy in cardiovascular disorders and β2-agonist treatment in respiratory disorders [bib_ref] A. β2-Adrenergic receptor genotype and survival among patients receiving β-blocker therapy after..., Lanfear [/bib_ref] [bib_ref] A. β1-and β2-adrenergic receptor gene variation, β-blocker use and risk of myocardial..., Lanfear [/bib_ref] [bib_ref] Arginine-16 β2 adrenoceptor genotype predisposes to exacerbations in young asthmatics taking regular..., Palmer [/bib_ref] [bib_ref] Adrenergic β2-receptor genotype predisposes to exacerbations in steroid-treated asthmatic patients taking frequent..., Basu [/bib_ref] [bib_ref] Association between genetic polymorphisms of the β2-adrenoceptor and response to albuterol in..., Martinez [/bib_ref]. However, not many studies have been conducted in the preterm labor treatment with β2-agonists, such as ritodrine. Since it is important to detect a correlation between drug phenotypes and single nucleotide polymorphisms (SNPs) studied, in order to utilize this correlation to benefit patients, the purpose of this study was therefore to evaluate the association between efficacy of ritodrine and ADRB2 gene polymorphisms in patients with preterm labor.
# Results
## Effects of patient characteristics on time to delivery
One hundred and fifty-four patients were enrolled, and electronic medical records of the patients were reviewed in Ewha Womans University Mokdong Hospital. Five patients who used ritodrine as a premedication for McDonald operation and eight patients who already had severe symptoms that led to preterm labor before hospital admission were excluded. Four patients who had to give up their babies because of the critical danger were excluded. Total seventeen patients were excluded by exclusion criteria, and one-hundred-thirty-seven patients were included. The mean maternal and gestational ages at admission were 31.6 ± 4.7 years and 29.5 ± 3.8 weeks, respectively. Seven patients had multiple gestations among 137 studied patients. The mean cervical dilation was 1.3 ± 1.2 cm.
The effects of demographic and clinical characteristics of patients on time to delivery are displayed in [fig_ref] Table 1: Effects of demographic characteristics on time to delivery [/fig_ref]. From univariate analysis, gestational ages at admission and Modified Bishop scores were statistically significant factors for time to delivery in preterm labor patients with ritodrine therapy (p < 0.001).
## Effects of genotypes on time to delivery
The genotypes and allele frequencies of studied SNPs of ADRB2 gene are shown in [fig_ref] Table 2: Effects of grouped genotypes in β-2 adrenergic receptor on the time to... [/fig_ref]. Minor allele frequencies ranged between 9.7% and 49.6%. The observed frequencies were consistent with Hardy-Weinberg equilibrium in all SNPs. The median time to delivery was 967.6, 741.0, and 287.1 h in patients with the wild-type homozygotes, heterozygotes, and mutant-type homozygotes in the Arg16Gly polymorphism, respectively. On the contrary, in the Gln27Glu polymorphism, longer time to delivery was found in patients with variant allele compared to those with the wild-type homozygotes. However, there was no significant association between the above two SNPs and pharmacologic responses of ritodrine treatment.
There was a linkage disequilibrium between c.252 G>A and c.523 C>A (r 2 = 0.984), and their effects on time to delivery were marginally significant (p < 0.1). Patients with wild-homozygotes showed prolonged time to delivery compared to those with variant alleles. The c.1053 G>C polymorphism significantly affected time to delivery; it took much shorter time to delivery in preterm labor patients with the GC, CC genotype than those with the GG genotype (p = 0.019, [fig_ref] Figure 1: Survival curve comparing GG group with the other two genotypes [/fig_ref]. Cox's proportional-hazards analysis demonstrated that maternal ages, modified Bishop scores, gestation ages at admission, and rs1042719 were significant predictors of time to delivery. Especially, the GC and CC carriers showed 64% decrease in time to delivery compared with the GG genotype patients in rs1042719 (p = 0.020) after adjusting other variables (maternal age and variables, which showed p < 0.1 from the univariate analysis) [fig_ref] Table 3: Multivariate analysis for the time to delivery [/fig_ref].
## Effects of genotypes on proportions of patients who remained undelivered
Proportions of patients who remained undelivered at 24 h, 48 h, and 10 days were not significantly different among the studied genotypes except for the Arg16Gly at 10 days; those with the Arg allele had approximately 1.6 times greater proportion compared to those with the variant-type homozygotes [fig_ref] Table 4: Effect of genotypes of β-2 adrenergic receptor on percentages of patients who... [/fig_ref].
# Discussion
Genetic contributions to preterm birth have been studied, recently focusing on genomic and proteomic approaches to diagnose and determine the mechanism of preterm labor. The genes or proteins investigated are involved in inflammatory reactivity or uterine contractility [bib_ref] Pharmacogenomics and individualized drug therapy, Eichelbaum [/bib_ref] [bib_ref] Activation of K + channels by ritodrine hydrochloride in uterine smooth muscle..., Hamada [/bib_ref]. ADRB2, which is expressed in smooth muscle cells of the myometrium, inhibits uterine contraction when it is activated. The association between ADRB2 polymorphisms and preterm labor has been focused on codons 16 and 27. The results were not consistent depending on the outcomes measured and study populations and did not favor a major impact of ADRB2 polymorphisms at positions 16 and 27 to the risk of preterm labor [bib_ref] Polymorphic variants of adrenoceptors: Pharmacology, physiology, and role in disease, Ahles [/bib_ref] [bib_ref] The genetic contribution towards preterm delivery, Adams [/bib_ref] [bib_ref] Genetic variation associated with preterm birth: A HuGE review, Crider [/bib_ref].
Studies of genetic polymorphisms suggest that it may account for 15%-30% of the variation in drug response [bib_ref] Pharmacogenetics of β-blockers, Shin [/bib_ref]. This study tried to approach from an aspect of different responses of tocolytic drugs due to genetic variances. Ritodrine binds to β2 adrenergic receptors on outer membrane of myometrial cell, activating adenylyl cyclase to increase the level of cAMP, which decreases intracellular calcium and leads to uterine contraction reduction [bib_ref] β2-Adrenergic receptor genotype and preterm delivery, Landau [/bib_ref]. Genetic variation in the structure of ADRB2 is a potentially significant source of variability in the response to β2-agonist drugs. Most commonly studied SNPs of ADRB2 gene are Arg16Gly (c.46 G>A) and Gln27Glu (c.79 C>G), non-synonymous variants, resulting in amino acid substitutions. The frequencies of the Gly16 and Glu27 allele were 49.6% and 9.7% in this study, which were consistent with the previous study [bib_ref] Association of the Gln27Glu polymorphism of the β2-adrenergic receptor with preterm labor, Ozkur [/bib_ref].
In terms of the effects of β2-agonist on preterm labor, the clinical outcomes after hexoprenaline administration to white women diagnosed with preterm labor were studied ADRB2 gene polymorphisms of Arg16Gly and Gln27Glu, which are two among five SNPs that we studied [bib_ref] Arg16 homozygosity of the β2-adrenergic receptor improves the outcome after β2-agonist tocolysis..., Landau [/bib_ref]. The results showed that pregnant women with Arg16 had a strong trend toward greater pregnancy prolongation, compared to the other genotype groups. The result was consistent with our results; patients with Arg16 alleles showed relatively prolonged time to delivery compared to those with the other genotype. Although, both studies narrowly missed to achieve statistical significance (p = 0.058 and 0.089, respectively).
In this study, patients with Arg16 alleles showed relatively prolonged time to delivery compared to those with the other genotype. Although Arg16 alleles did not show significant prolongation of time to delivery, significantly greater proportions of patients who remained undelivered at 10 days were found in the Arg16 allele carriers compared to those with Gly16 allele homozygotes. This result was attributable to not only the protecting effect of the Arg16 allele but also the receptor downregulating effect of Gly16 allele. An in vitro study showed that the Gly16 allele resulted in enhanced agonist-promoted down-regulation. The in vitro study also reported that the Glu27 allele was resistant to receptor down-regulation [bib_ref] β2-Adrenergic receptor pharmacogenetics, Liggett [/bib_ref]. However, significant association between the Gln27Glu polymorphism and time to delivery in preterm labor patients with ritodrine therapy was not found in this study.
Gln27Glu is in high linkage disequilibrium with Arg16Gly [bib_ref] Complex promoter and coding region β2-adrenergic receptor haplotypes alter receptor expression and..., Drysdale [/bib_ref]. Therefore, Gln27Glu could be considered as a cofactor of Arg16Gly effects. To obtain complementary information regarding the effectiveness of ritodrine therapy, the haplotype analysis of the two genotypes was carried out. Although patients with both Gly16 and Gln27 homozygotes (median time: 215.7 h) showed shorter time to delivery than those with the other haplotypes (median time: 828.0 h), significant association was not found (p = 0.285).
Strong linkage disequilibrium between rs1042717 and rs1042718 was found in this study, consistent with earlier studies [bib_ref] Genotypes and haplotypes of β2-adrenergic receptor and parameters of the metabolic syndrome..., Park [/bib_ref] [bib_ref] Polymorphisms in the ADRB2 gene and graves disease: A case-control study and..., Chu [/bib_ref]. Significantly longer time to delivery in GG allele carrier of rs1042719 was obtained after adjusting other covariates. Unlike rs1042713 and 1042714, rs1042717, 1042718, and 1042719 are synonymous polymorphisms, which were not expected to change the β2-adrenoreceptor activity. The effect of synonymous SNP in the ADRB2 gene on the ritodrine therapy in this study was possibly attributable to the linkage disequilibrium with other SNPs, which have functional activities [bib_ref] A "silent" polymorphism in the MDR1 gene changes substrate specificity, Kimchi-Sarfaty [/bib_ref]. In addition, a recent study showed that synonymous coding SNPs can affect the translation rate of mRNA, resulting in the change of protein amount produced and the post-translational modification of the protein [bib_ref] Differential arginylation of actin isoforms is regulated by coding sequence-dependent degradation, Zhang [/bib_ref]. Another possibility is that it interferes with an exon-splicing enhancer to affect RNA processing [bib_ref] Discovery of novel flavin-containing monooxygenase 3 (FMO3) single nucleotide polymorphisms and functional..., Koukouritaki [/bib_ref].
Although SNP detection and genotyping studies have shown to explain and diagnose many diseases and to describe the variation in drug responses, most findings have not been replicated. This is because clinical outcomes are affected by other factors as well as genetic predisposition. Especially, the long-term efficacy and therapeutic usefulness of β2-agonist drugs have been questioned in many studies, and the limitation of the therapeutic value of β2-agonist drugs was attributable to the desensitization of β-adrenergic receptors in the myometrium by agonist-stimulation [bib_ref] Decresaes myometrial β-adrenoceptors in women receiving β2-adrenergic tocolytic therapy: Correlation with lymphocyte..., Michel [/bib_ref] [bib_ref] Self-limitation of intravenous tocolysis with β2-adrenergic agonists is mediated through receptor G..., Frambach [/bib_ref]. In the animal study with preterm labor, ritodrine initially reduced labor contractions but the effects decreased within 16 h [bib_ref] Tocolytic therapy with fenoterol induces selective down-regulation of β-adrenergic receptors in human..., Engelhardt [/bib_ref]. The reduced effects were explained by a reduced cAMP response to β2-agonist drugs and by decreased number of β-adrenergic receptors [bib_ref] Decresaes myometrial β-adrenoceptors in women receiving β2-adrenergic tocolytic therapy: Correlation with lymphocyte..., Michel [/bib_ref] [bib_ref] Self-limitation of intravenous tocolysis with β2-adrenergic agonists is mediated through receptor G..., Frambach [/bib_ref]. Furthermore, studies showed that neither the Arg16Gly nor the Gln27Glu polymorphism affected the extent of agonist-induced desensitization, but the Glu27 homozygotes exhibited a slower desensitization compared to the other genotypes [bib_ref] The effect of common polymorphisms of the β2-adrenergic receptor on agonist-mediated vascular..., Dishy [/bib_ref] [bib_ref] Genotype-dependent time course of lymphocyte β2-adrenergic receptor down-regulation, Bruck [/bib_ref].
Another consideration for clinical application of genomic studies is that failure to achieve statistical significance does not necessarily mean clinical insignificance. In this study, most SNPs shortened the time to delivery by more than 50% but statistical significance was not found. This was possibly due to the small sample size, resulting in underpowered study. Given the single-center and the relatively small sample size, our hypothesis requires further independent validation using multi-center and large sample size.
# Materials and methods
## Study patients
This prospective follow-up study was conducted at Ewha Womans University Mokdong Hospital from January 2010 to February 2013, approved by Ethics Committee of the Ewha Womans University Mokdong Hospital Institutional Review Board. (IRB No.: 217-1-26, 6 January, 2010) Patients were eligible for participation if they met following criteria: preterm labor with intact membrane, gestational age of 20 to 36 weeks, ≥18 years of age, uterine contractions with a frequency of 3 per 10 min with cervical change, and provision of written informed consent. Exclusion criteria were severe pre-eclampsia, placenta abruptio, fetal distress, severe oligohydroamnios, fetal/placental/ amniotic abnormalities, placenta previa, severe spontaneous premature rupture of membrane, and women whose continuation of pregnancy would be dangerous for them. Patients treated with tocolytics other than ritodrine were excluded. In addition, cases in which ritodrine was used for preventing uterine contraction during the Mcdonald operation were excluded.
## Drug administration
Ritodrine (Lavopa ® ; JW Pharmaceutical, Seoul, Korea), was given as an intravenous infusion at an initial rate of 0.05 mg/min and increased by 0.05 mg/min every 10 min, until the desirable uterine response was obtained. Intravenous treatment was discontinued during uterine quiescence. Patients who achieved uterine quiescence received maintenance therapy via an infusion of 0.05 mg/min for 12 to 48 h.
## Outcomes and data collection
The primary end point was time (hour) to delivery. Secondary endpoint was proportion of patients who remained undelivered at 24 h, 48 h, and 10 days. Patients' data, including maternal age, gestational age at admission, type of gestation, cervical dilation, Modified Bishop score, and mode of delivery, were collected. The paper-based and electronic medical records of patients were reviewed.
# Genotyping methods
Genomic DNAs were extracted from EDTA-blood samples by usage of QIAamp DNA Blood Mini Kits (QIAGEN GmbH, Hilden, Germany) according to standard procedures recommended by a manufacturer. The following SNPs were genotyped of ADRB2 gene using The TaqMan ® allelic discrimination technique by RT-PCR system (ABI 7300, Applied Biosystem, Carlsbad, CA, USA): rs1042713 (c.46 A>G, Arg16Gly), rs1042714 (c.79 C>G, Gln27Glu), rs1042717 (c.252 G>A, Leu84Leu), rs1042718 (c.523 C>A, Arg175Arg), and rs1042719 (c.1053 G>C, Gly351Gly).
# Statistical analysis
Continuous variables were compared by the Student's t-test; when there were more than two comparison groups, a one-way ANOVA was used. If the variables were not normally distributed, as determined by the one-sample Kolmogorov-Smirnov and Levene tests, additional Mann-Whitney and Kruskal-Wallis tests were used. The Chi-square test was used to compare categorical variables. The interval from start of treatment to delivery was analyzed with Kaplan-Meier survival data analysis method (log-rank test). Cox's proportional-hazards model was used for exploratory multivariate analysis for the interval from start of treatment to delivery. All the analyses were based on two-tail statistics. The data were analyzed using Statistical Package for Social Sciences Version 17.0 for Windows (SPSS 17.0K, SPSS Inc., Chicago, IL, USA). A p-value of less than 0.05 was considered statistically significant.
# Conclusions
The polymorphisms of ADRB2 gene could affect ritodrine therapy in patients with preterm labor. Given the burden of preterm labor, this pharmacogenomic study could have the potential to improve the management of this disorder by accounting for some of the inter-individual variability in pharmacologic response of ritodrine therapy. Since the study population is uni-ethnic, further study is required on other populations.
[fig] Figure 1: Survival curve comparing GG group with the other two genotypes (GC, CC group, p = 0.019) of ADRB2 rs1042719 by Kaplan-Meier survival analysis. [/fig]
[table] Table 1: Effects of demographic characteristics on time to delivery. [/table]
[table] Table 2: Effects of grouped genotypes in β-2 adrenergic receptor on the time to delivery. There were a total of 8 failures in genotyping 5 SNPs of 137 patients. [/table]
[table] Table 3: Multivariate analysis for the time to delivery.Heterozygote and mutant homozygote vs. wild-type homozygotes; Adjusted for all factors of which p-value was less than 0.1 from univariate analysis. [/table]
[table] Table 4: Effect of genotypes of β-2 adrenergic receptor on percentages of patients who remained undelivered.Proportion (%) p Value Proportion (%) p Value Proportion (%) p Value [/table]
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Characteristics of Wind Velocity and Temperature Change Near an Escarpment-Shaped Road Embankment
Artificial structures such as embankments built during the construction of highways influence the surrounding airflow. Various types of damage can occur due to changes in the wind velocity and temperature around highway embankments. However, no study has accurately measured micrometeorological changes (wind velocity and temperature) due to embankments. This study conducted a wind tunnel test and field measurement to identify changes in wind velocity and temperature before and after the construction of embankments around roads. Changes in wind velocity around an embankment after its construction were found to be influenced by the surrounding wind velocity, wind angle, and the level difference and distance from the embankment. When the level difference from the embankment was large and the distance was up to 3H, the degree of wind velocity declines was found to be large. In changes in reference wind velocities around the embankment, wind velocity increases were not proportional to the rate at which wind velocities declined. The construction of the embankment influenced surrounding temperatures. The degree of temperature change was large in locations with large level differences from the embankment at daybreak and during evening hours when wind velocity changes were small.
# Introduction
When a highway is constructed in a mountainous area, highway embankments and tunnels may be introduced. Highways constructed in mountainous areas using such methods may damage agricultural products by disturbing the natural sunlight and ventilation of an area. When an artificial structure such as an embankment is constructed in a "fruit" farming area, the disturbance of natural airflow can cause the temperature of the area to change, which can result in damage such as the withering away of fruit trees, reduced crop yields, and delays in blooming, all of which reduce the quality of the crops. Although wind corridors can be installed around embankments to prevent the damage of cold weather caused by cutting off the airflow, they have not been particularly effective. Most of the cold-weather damage to fruit trees in areas with highway embankments occurs in the spring when the wind is weak. This is because most cold-weather damage is caused by poor airflow. Particularly, when slopes are being constructed on valley-shaped open land, the free flow of air is blocked by highway embankments, and the temperature in the area becomes lower than that in other neighboring areas, enhancing the cold-weather damage. Airflow changes in sloped areas are more complicated and more diversified due to topographic effects.
There are many phenomena unique to different topographies, such as gusting and wind velocity increases and decreases caused by covering effects. Wind velocity is increased on a slope, and it may be increased by certain other topographic effects of the land. Many studies have examined the increase in wind velocities in mountainous, valley, and sloped areas. Both Jackson and Hunt [bib_ref] Turbulent wind flow over a low hill, Jackson [/bib_ref] and Mason and Sykes [bib_ref] Flow over an isolated hill of moderate slope, Mason [/bib_ref] studied the effects of wind velocity increases in lower mountainous areas without separation phenomena. Bowen [bib_ref] The prediction of mean wind speed above simple 2D hill shapes, Bowen [/bib_ref] studied wind velocity in simple two-dimensional mountainous areas. Tayor and Lee [bib_ref] Simple guidelines for estimating wind speed variations due to small scale topographic..., Tayor [/bib_ref] proposed an algorithm to forecast wind velocity increases at the peak of a mountainous area. Most studies have focused on the distribution of wind velocities under various warm current conditions in mountainous areas , Neff and King [bib_ref] Observations of complex-terrain flows using acoustic sounders: experiments, topography, and winds, Neff [/bib_ref] , Finardi et al. [bib_ref] An assessment of mixing-length closure schemes for models of turbulent boundary layers..., Finardi [/bib_ref] , Booij et al. [bib_ref] A third-generation wave model for coastal regions 1. Model description and validation, Booij [/bib_ref] , and Vosper et al. [bib_ref] Measurements of the near-surface flow over a hill, Vosper [/bib_ref]. Miller and Davenport [bib_ref] Guidelines for the calculation of wind speed-ups in complex terrain, Miller [/bib_ref] and Li et al. [bib_ref] Comparison on topographical speed-up effects on wind flow provided by main load..., Li [/bib_ref] performed comparative analyses on wind velocity increases in complex mountainous areas by considering surface roughness suggested in major loading criteria and surrounding geographical features. In addition, they emphasized surface roughness and surrounding air current conditions when predicting wind velocity increases. Weng et al. [bib_ref] Guidelines for airflow over complex terrain: model developments, Weng [/bib_ref] proposed guidelines on air currents in complex geography by considering geographical features and surface roughness. Svoboda andČermák [bib_ref] Vertical profiles of wind speed over a mountain ridge, Svoboda [/bib_ref] measured the wind velocities and their distribution in the ridges of the Erzgebirge Mountains using Doppler Sodar observations. Chock and Cochran [bib_ref] Modeling of topographic wind speed effects in Hawaii, Chock [/bib_ref] performed a wind tunnel test to investigate the rate of the wind increase phenomenon on an island with a varied topography and proposed an experimental model regarding the peak and wind velocity increase that could be applied to the design of field structures.
However, highway embankments influence the lower currents at the bottom of a slope. There have not been sufficient studies carried out on airflow near an artificial structure such as a highway embankment. As fruits grown in the bare ground at the bottom of a slope are sensitive to temperature and wind velocity, wind velocity and temperature should be evaluated before constructing roads on the slope. In this study, wind velocity and temperature changes before and after the construction of highway embankments on valleyshaped open land were investigated. For changes in wind velocity, a wind tunnel test was performed using models. In the wind tunnel test, a model was used to identify the wind velocity change before and after the construction of highway embankments. The correlation between the wind velocity and temperature near highway embankments was identified when the field experiment in the highway embankment's neighboring areas was conducted.
## Location and study method
In the test site, fruit farms were distributed around an area comprised of a 1.5 km embankment in a highway construction section. The highway embankments, located at 36 ∘ 3.4 N and 140 ∘ 7.5 (E), and their surrounding areas are shown in [fig_ref] Figure 1: Topographic map of area surrounding embankment [/fig_ref]. Before the construction of the embankments, air could naturally flow down to the bottom of the mountain. However, it seems that the construction of the embankments affected the airflow. To evaluate wind velocity and temperature change in the areas surrounding the highway embankments, two types of tests were executed. First, by making a miniature land model, a wind tunnel test was performed to identify wind velocity changes in survey points before and after the construction. Second, a field experiment was performed to identify the correlation between temperature and wind velocity changes in the fruit farming area after implementation of the embankment.
## Wind tunnel test
## Experimental model.
To identify the flow of air near the highway embankments, a wind velocity test was performed upon a 1/150 scaled land model. The land model for the wind tunnel was made of Styrofoam, and an aluminum bar was installed so that anemometers could be installed to measure wind velocity. The turbulent boundary layer wind tunnel device was a vertical circulation closed-circuit type, and the sectional size of the tunnel was 12 m in width, 2.5 m in height, and 40 m in length. shows the experimental land model installed inside the wind tunnel. To identify wind velocity changes in highway embankments, multichannel anemometers (System 6242 Model 1560) were used. The experiment was performed to identify changes in wind velocity according to the level difference in the surrounding topography before and after the construction of highway embankments with a certain initial wind velocity. To identify changes in wind velocity below the embankment, a total of 19 points were selected, as there was a difference in the levels from the southern side and northern side of the embankment. The anemometer was installed only in the southern direction. As the southern area was larger than the northern area, it was used as an orchard. Wind velocity tests were conducted in five sites just below the embankment and in 14 sites at random distances from the embankment. The tested wind angles were limited to those of the winds blown from the northern and southern directions of the embankment. The wind velocity tests were performed on 10 wind angles including each set of four wind angles with a 22.5 ∘ gap between the NW-NE wind angle and the SW-SE wind angle. presents the wind angles in the wind tunnel test. The anemometer that measured reference wind velocities was installed above the road with the embankment. The heights of 19 measurement sites and the reference anemometer were fixed at 10 mm (fullscale height was 1.5 m).
## Wind velocity measurement
Results. Three reference wind velocities were used in measuring wind velocities: 3 m/s, 5 m/s, and 7 m/s. The reference wind velocities were based on the wind velocities measured by the anemometer on the embankment road. This test examined wind velocity changes by measurement site according to changes in the reference wind velocities in the surrounding area before and after the construction of the embankment. shows the level difference based on the measurement sites to measure wind velocities around the embankment and the height of the embankment road. The adjacent area below the embankment had an average level difference of −8.5 m. Based on the central point of the embankment, the left area had the largest level difference of −11 m, and the right area had a level difference of −5.9 m. [fig_ref] Figure 4: Outline of measurement of wind velocity [/fig_ref] shows the outline of the wind velocity measurements by reference wind velocity and measurement site. [fig_ref] Figure 5: Distribution of wind velocities by measurement site according to wind angle changes... [/fig_ref] shows the distribution of wind velocities by measurement site according to wind angle changes in the area to the right of the embankment. Wind velocity changes by measurement site were found to vary compared to the reference wind velocities according to wind angle changes. However, the wind velocity of the southeastern position as the valley wind on the land was at most 60% smaller than the wind velocities measured from other wind directions. After the construction of the embankment road, large declines in wind velocity were shown compared to the reference wind velocities in all measured wind directions except some northern directions. The wind directions (N and NNW) with small wind velocity changes before and after the construction of the embankment were found in the sites with lower embankment heights than the other sites. This study examined wind velocity changes according to increases in the reference wind velocities before and after the construction of the embankment. The wind angles from some northern directions (N, NNW, and NW) before and after the construction of the embankment showed that the rates of decline of wind velocities after construction were small at less than 20% regardless of the measurement location or wind velocity. A smaller distance between the measurement site and the embankment and an increase in the reference wind velocity resulted in a corresponding larger degree of wind velocity decline. This study examined wind velocity changes compared to the reference wind velocities according to the level difference between the embankment's height and the measurement site. In the case of Measurement Site 3, it was −13.6 m under the embankment road. After its construction, wind velocity changes were 1 or below in all wind velocities. It was confirmed that the rate of decline of wind velocities was being influenced by the level difference from the embankment. [fig_ref] Figure 6: Distribution of wind velocities by measurement site according to wind angle changes... [/fig_ref] shows wind velocity changes by measurement site according to wind angle changes in the area to the left of the embankment. The left area contained many areas that were over 50% higher in terms of the average level difference. The left area was also being influenced by the measurement sites and wind angles in the degree of wind velocity changes compared to the reference wind velocities before and after the construction of the embankment. Measurement Site 5 located just below the embankment had a level difference of −11.5 m from the embankment road and showed a large degree of wind velocity decline with over 70% after the construction of the embankment at a reference wind velocity of 3 m/s. However, Measurement Sites 9, 14, and 15 exhibited little wind velocity changes compared to the reference wind velocities regarding the wind angles of the southern direction before and after the construction of the embankment. This is probably because these sites had larger level differences than the equivalent right sites. It was confirmed that the wind velocity changes around the embankment were largely influenced by the distance and level difference from the embankment. [fig_ref] Figure 7 6: ∘ , maximum temperature of 21 [/fig_ref] shows wind velocity changes by distance from the embankment according to wind angle changes. Before the construction of the embankment, wind velocity changes by distance were shown to be consistent without large influences from wind angles. However, after the construction of the embankment, wind velocity changes compared to the reference wind velocities according to the distance from the embankment were confirmed to be influenced by wind angles. In wind velocity changes by the measurement distance of the wind angles SSW and SW, the site that was 3 ( = the embankment's height) away from the embankment showed a decline of wind velocity ratios of up to over 60% compared to the site 1.5 away from the embankment regardless of wind velocity changes. However, in the wind angle NNW blown from the embankment's north, there were no wind velocity changes by distance. Wind velocity changes by distance from the embankment were influenced by wind angles. [fig_ref] Figure 8: Overall wind velocity distribution chart within location before and after construction of... [/fig_ref] shows the wind velocity distribution of the area surrounding the embankment when the wind was blowing from SSW at 3 m/s. [fig_ref] Figure 8: Overall wind velocity distribution chart within location before and after construction of... [/fig_ref] below the wind velocity distribution shows the field topography by color. Areas with lower altitudes The Scientific World Journal are shown in black, and higher altitudes are shown in red. Before inserting the model slope, the wind velocity was distributed according to topography. Therefore, the left area, which had higher topography, always had at least 2 m/s of wind velocity. In the lower level, there was always at least 1.35 m/s of wind velocity.
However, when highway embankments were constructed, the right area with its lower topography had a more than 55% wind velocity reduction, which reduced the wind velocity to less than 1 m/s. There was no significant reduction in wind velocity in the left area with a smaller level difference.
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## Field experiment
To identify the correlation between the surface wind velocity and the temperature change in the area of highway embankments, a field experiment was performed. shows the distance between the meteorological observatory and the field experiment site (8.6 km in a straight line from the measured points). The field experiment was conducted based on an average temperature of 5. [fig_ref] Figure 1: Topographic map of area surrounding embankment [/fig_ref] shows the location of the field experiment site. To identify changes in wind velocity and temperature according to the height of the embankment, anemometers were installed at the highest and lowest points. Five points between the two anemometers were selected as temperature measure points. The temperature change was recorded for 18 days, and the average temperature data measured every 5 minutes was automatically saved. The measurement range of the temperature sensor (HOBO Pro v2 Tem/RH Data Logger) was −40-70 ∘ C, and the measurement range of the wind velocity sensor was 0.5-50 m/s. [fig_ref] Figure 1: Topographic map of area surrounding embankment [/fig_ref] compares the temperatures (average, maximum, and minimum) and wind velocities between the data recorded in the meteorological observatory and that measured in the field experiment during the 18-day experimental period. The weather station was 8.6 Km away from the field measurement location in straight-line distance, but their average temperatures were consistent. However, the number of days when a minimum temperature of below 0 ∘ C was observed was 9 days according to the meteorological observatory but 15 days in the field experiment, which means that the fieldmeasured points had six more days that showed a minimum temperature of below 0 ∘ C. When the average temperature in the meteorological observatory was −4.1 ∘ C, it was −9.1 ∘ C at the field experiment site. For the average wind velocity distribution, a wind velocity of 1.1 m/s-2 m/s was shown for eight days in the field, while it was shown for only two days at the meteorological observatory. A wind velocity higher than 3 m/s was shown for three days in the field experiment and nine days at the meteorological observatory. The wind velocity was lower in the field-measured points than at the meteorological observatory. When comparing meteorological data between the meteorological observatory and the field experiment site during the experimental period (18 days), it was found that higher temperatures and lower wind velocities were observed more often at the field experiment site, although the highest recorded temperatures were almost identical. [fig_ref] Figure 1: Topographic map of area surrounding embankment [/fig_ref] shows the average wind velocity and temperature at points (1.2 m and −11.5 m from the embankment) plotted against time. It was found that the temperature dropped below 0 ∘ C as the wind velocity rapidly decreased before 6 am and after 18 pm.
The lowest point in the site (Temperature Measure Point 1) showed a 2 ∘ C lower temperature than the other point of the same height on the embankment (Temperature Measure Point 6). Temperature and wind velocity increased from 8 am and reached a peak at 14 pm. Afterwards, both the temperature and wind velocity decreased. However, the temperatures and wind velocities at points lower than the height of the embankment were up to 40% lower than those at points higher than the embankment. From these results, it was confirmed that both temperature and wind velocity were affected by filling in at the field experiment site. In general, temperature distributions by height do not yield large temperature deviations by height on cloudy days due to small amounts of radiation. However, they show large temperature deviations by height on clear and windless days. While the temperatures of low-level sites installed with the embankment were measured at lower levels than those of high-level sites at dawn at below-zero temperatures and in the evening, they were measured at higher levels at noon when the temperature rose. In other words, a temperature-reversal phenomenon was observed.
This temperature-reversal phenomenon is shown in [fig_ref] Figure 1: Topographic map of area surrounding embankment [/fig_ref] , which shows a graph of time averages during the measurement period. In the measured data, the temperature in lower areas was 2.0 ∘ C lower than in higher areas at night, but it was also 3.5 ∘ C higher in the daytime. [fig_ref] Figure 1: Topographic map of area surrounding embankment [/fig_ref] shows 24 hours' worth of data measured at survey points on rainy days and the days prior to rainy days. In the daytime before rainy days, there was a clear temperature-reversal phenomenon in lower areas. The temperature was below zero in the dawns and evenings and above zero in the afternoons. However, during rainy days, all survey sites showed tiny temperature differences between the day and night of less than 1 ∘ C.
## Relationship between wind velocity and temperature change
The distribution chart of wind velocities and temperatures following the construction of the embankment was examined. [fig_ref] Figure 1: Topographic map of area surrounding embankment [/fig_ref] shows the distribution chart of hourly wind velocities and temperatures by experiment site. Based on geographical characteristics, 18 days' worth of data from a high-level site (+1.2 m based on the embankment site) and
## 12
The Scientific World Journal a low-level site (−13.6 m based on the embankment site) were used. To understand the characteristics of wind velocities and temperature changes, an hourly analysis (18 pm-6 am and 6 am-18 pm) was performed. Wind velocity changes at dawn and during evening hours were very low at below 0.3-0.5 m/s. The low-level site (temperature 1) below the embankment showed temperature changes in the range of 0 to −4 ∘ C, while the high-level site showed temperature changes ranging from 0.4 to −0.4 ∘ C. The low-level site revealed a larger range of temperature changes than the high-level site. During the hours when the measured wind velocity was very low at 0.5 m/s, the low-level site recorded below-zero temperatures in all temperature ranges. The low-level site's minimal temperature of −4 ∘ C showed a temperature difference over ten times that of the high-level site within the same range of wind velocities. During the morning and afternoon hours when the wind velocity was measured at 2.4 m/s or lower, the difference between the maximum and minimum temperatures in the low-level site was 10 ∘ C. However, the difference in the highlevel site was 5 ∘ C. Regarding the characteristics of hourly temperatures, it was confirmed that the embankment reduced the wind velocity and lowered the temperature to the belowzero range. It was also determined that stagnant regions without wind velocity changes due to the embankment influenced the temperature.
# Conclusion
The results of this study regarding wind velocity and temperature changes caused by the embankment around a highway constructed on a sloped topography are as follows.
Wind velocity changes around the embankment were influenced by surrounding wind velocities, wind angles, the level differences of surrounding areas according to the embankment's height, and the distance of areas from the embankment. Wind velocity changes were evaluated in various terms according to the measurement site. However, a lower reference wind velocity exhibited a corresponding larger rate of decline of wind velocities. In addition, in terms of wind angle changes, the velocities of winds blown from sloped and valley-shaped areas decreased by up to over 60% after the construction of the embankment. In addition, the rate of decline of wind velocities due to the level difference of surrounding areas according to the embankment's height was found to be largest in the area with the largest level difference from the central part of the embankment. Wind velocity changes by the distance from the embankment exhibited an increase in the decline range of wind velocities up to the distance of 3 . Field measurements were conducted to determine wind velocity and temperature changes after the construction of the embankment. The results of the field measurements also confirmed wind velocity changes according to the embankment's height and level difference. In the central part of the embankment, the lowest wind velocity was measured, whereas the degree of wind velocity change was found to be small. The results of the wind tunnel test were in line with the general tendency. The site with small wind velocity changes (below the embankment) recorded lower temperatures than the higher site. Temperature changes in the evening and at dawn when low wind velocities were measured were larger compared to other hours. After the construction of the embankment, temperatures also dropped along with wind velocities.
[fig] Figure 1: Topographic map of area surrounding embankment. [/fig]
[fig] Figure 2, Figure 3: Land model installed inside wind tunnel. Wind angles and measurement points. [/fig]
[fig] Figure 4: Outline of measurement of wind velocity. [/fig]
[fig] Figure 5: Distribution of wind velocities by measurement site according to wind angle changes in area to right of embankment. [/fig]
[fig] Figure 6: Distribution of wind velocities by measurement site according to wind angle changes in area to left of embankment. [/fig]
[fig] Figure 7 6: ∘ , maximum temperature of 21.4 ∘ , minimum temperature of −4.1 ∘ , Wind velocities per minute by embankment's distance according to wind angle changes. velocity of 3.4 m/s in March (as observed in the nearest meteorological observatory). In the field experiment, wind velocity and temperature distribution were identified focusing on the lowest point (−11.5 m) and the highest point (1.2 m) of the embankment. [/fig]
[fig] Figure 8: Overall wind velocity distribution chart within location before and after construction of embankment (wind angle = SSW). [/fig]
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Novel P397S MAPT variant associated with late onset and slow progressive frontotemporal dementia
Mutations in the MAPT gene cause frontotemporal dementia with tau deposits. We report the novel p.P397S MAPT variant in eight subjects from five apparently nonrelated families suffering from frontotemporal dementia with autosomal dominant pattern of inheritance. In silico analysis reported conflicting evidence of pathogenicity. The segregation analysis support that this variant is likely pathogenic. The mean age at onset (61.4 years) and mean disease duration (13.9 years) of these subjects and their affected relatives were significantly higher compared with our series of p.P301L MAPT mutation carriers. These findings suggest that p.P397S variant could be a new MAPT mutation associated with a less aggressive phenotype than other MAPT mutations.
# Introduction
Mutations in the microtubule-associated protein tau (MAPT) gene in chromosome 17 were described in 1998 as a cause of frontotemporal lobar degeneration (FTLD) with tau pathology. [bib_ref] Tau is a candidate gene for chromosome 17 frontotemporal dementia, Poorkaj [/bib_ref] [bib_ref] Association of missense and 5'-splice-site mutations in tau with the inherited dementia..., Hutton [/bib_ref] To date, nearly 50 pathogenic MAPT mutations have been reported. [bib_ref] Locus-specific mutation databases for neurodegenerative brain diseases, Cruts [/bib_ref] Clinically, FTLD related to MAPT mutations may affect behavior, language, memory, and executive functions. Parkinsonism has also been reported in some cases. Most MAPT mutations are characterized by early onset (mean 47.9 years) and a rapidly progressive clinical deterioration with a mean age at death of 58.7 years. [bib_ref] Loss of progranulin function in frontotemporal lobar degeneration, Cruts [/bib_ref] We describe the clinical phenotype of a novel exon 13 MAPT varianta Proline to Serine substitution at codon 397 (p.P397S)in eight subjects with FTLD with an autosomal dominant pattern of inheritance from five apparently nonrelated families. We hypothesize that this variant could be causative of FTLD. In addition, we compare the clinical phenotype of these cases with our series of the most frequent (p.P301L) MAPT mutation. [bib_ref] Frontotemporal dementia caused by the P301L mutation in the MAPT gene: clinicopathological..., Borrego-Ecija [/bib_ref] Methods Subjects were recruited from the Genetic counseling program for familial dementias (PICOGEN program) at the Hospital Cl ınic de Barcelona. [bib_ref] PICOGEN: five years experience with a genetic counselling program for dementia, Fortea [/bib_ref] A detailed clinical history and neurological evaluation of all subjects were performed. We reviewed the information available from all the affected cases in the pedigrees. The diagnosis of behavioral variant frontotemporal dementia (bvFTD) was performed following the current diagnostic criteria. [bib_ref] Sensitivity of revised diagnostic criteria for the behavioural variant of frontotemporal dementia, Rascovsky [/bib_ref] Cerebrospinal fluid (CSF) analysis for Alzheimer's disease (AD) biomarkers was measured with INNOTEST ELISAs following manufacturer's instructions (Fujirebio, Ghent, Belgium). Our laboratory normal reference values for amyloid beta 42 (AB42), total tau (t-tau), and phosphorylated tau at threonine 181 (p-tau) are >660, <385, and >65 pg/mL, respectively.
## Ethics
This research was performed according to the guidelines of the Declaration of Helsinki. The participants provided written informed consent for genetic testing and publication of relevant findings. The study was approved by the Hospital Cl ınic Ethics committee.
# Genetic analysis
Genetic screening for MAPT (exons 1 and 9-13) and progranulin (GRN) (exons 1-13) or serum progranulin levels was performed in the eight subjects as previously described. [bib_ref] Serum progranulin levels in patients with frontotemporal lobar degeneration and Alzheimer's disease:..., Antonell [/bib_ref] The MAPT haplotype (H1/H2) was determined studying the SNP rs1800547 and APOE genotype with two genotyping assays (rs429358 and rs7412), using TaqMan genotyping technologies (Life Technologies, Carlsbad, CA). C9ORF72 GGGGCC hexanucleotide repeat expansion was tested with a repeat-primed PCR. Subject II.II.VIII was examined by exome sequencing using MedExome (Roche, Basel, Switzerland) in an Illumina NextSeq500.
We searched for functional information at ENSEMBL database (https://www.ensembl.org), where the variant was described as rs1295855402, and with algorithms that predict whether an amino acid substitution affects the protein function such as SIFT, Polyphen-2, REVEL, Mutation Assessor, CADD, and MetaLR.
# Statistical analysis
Mean age at onset, disease duration, and age at death were calculated considering together subjects with proven variant and their affected relatives. Results in p.P397S carriers were compared with our series of p.P301L using Fisher's exact test. Disease duration was analyzed using Kaplan-Meier estimator and compared with Log-rank test. The analyses were performed using the SPSS Statistics Version 20.0 IBM Corp, Chicago, IL), and the level of significance was established at a P level of 0.05 (twosided).
# Results
## Clinical phenotype
We include here the description of several representative cases.
Family I Subject I.III.IV developed behavioral disinhibition, sweet food preference, deficits in executive tasks, and semantic language impairment at the age of 60 meeting criteria for bvFTD. MRI showed severe medial, lateral, and polar bitemporal atrophy. Currently, after 17 years of disease, she is in a stage of moderately severe dementia with severe semantic impairment scoring 5/30 at Boston Naming Test (BNT), but preserved motor functions. She had an autosomal dominant family history of dementia on her mother, aunt, grandmother, and three siblings [fig_ref] Figure 1: Pedigree of the five reported families [/fig_ref]. All of them developed cognitive and behavioral dysfunction starting from 50 to 77 years old and showing slow disease progression. Due to this family history of dementia genetic exam was performed in subject I.III.IV and one of her brothers (subject I.III.III) revealing the presence of the p.P397S MAPT variant in both.
## Family ii
Subject II.II.IX developed socially inappropriate behavior at the age of 64. Neuropsychological evaluation revealed short-term memory and semantic language impairment (BNT 35/60). The MRI demonstrated moderate bitemporal atrophy [fig_ref] Figure 2: MRI of subject III [/fig_ref]. The CSF core biomarkers analysis showed normal levels of AB42 (1308 pg/mL), but increased levels of t-tau (414 pg/mL) and p-tau (87 pg/ mL). He had a family history of late-onset dementia on his father, and three siblings [fig_ref] Figure 1: Pedigree of the five reported families [/fig_ref]. The genetic study demonstrated the presence of the p.P397S variant in the MAPT gene. Currently, after 8 years of disease duration, he still is in a moderate stage of dementia, scoring 24 at the MMSE and 102 at the Revised Cambridge Behavioral Inventory (CBI-R).
Subject II.II.VIII was a sibling of subject II.II.IX. He initially developed episodic and semantic memory loss and behavioral disinhibition at the age of 67. The CT scan showed bitemporal atrophy and the CSF exam presented decreased levels of AB42 (437 pg/mL) and increased t-tau (543 pg/mL) and p-tau (80 pg/mL), so AD diagnosis was established. During the follow-up, the patient developed severe apathy, loss of empathy, hyperorality, and executive dysfunction, highly suggestive of bvFTD. He also presented mild rigid-akinetic parkinsonism. Currently, after 8 years of disease progression, he scores 25 at the Mini Mental State Examination (MMSE) and he is independent for basic activities of daily living and most of the instrumental activities. The genetic test confirmed the presence of the p.P397S variant, consequently the final diagnosis of genetic bvFTD with possible concomitant AD was established.
The p.P397S MAPT variant was not found in subject II.II.VII, an asymptomatic 68-year-old sibling.
Family III Subjects III.II.VI and III.II.VII were dizygotic twins. Their mother developed dementia at the age of 65. Another sibling presented clinically with behavioral impairment dysfunction strongly suggestive of bvFTD [fig_ref] Figure 1: Pedigree of the five reported families [/fig_ref]. Subject III.II.VI presented with disinhibition, apathy, and ritualistic behaviors at the age of 63. Neuropsychological evaluation revealed impairment in memory and naming. MMSE score was 19 at the age of 69. The MRI showed severe medial bitemporal atrophy [fig_ref] Figure 2: MRI of subject III [/fig_ref] presented oculomotor impairment. All subjects presented bitemporal atrophy with relative frontal lobe preservation [fig_ref] Figure 2: MRI of subject III [/fig_ref]. The frequency of these clinical symptoms does not differ from our published series of p.P301L mutation carriers. [bib_ref] Frontotemporal dementia caused by the P301L mutation in the MAPT gene: clinicopathological..., Borrego-Ecija [/bib_ref] Subjects with the p.P397S MAPT variant presented a significantly older age at onset than the patients with the p.P301L MAPT mutation (61.3 years vs. 53.5 years; P = 0.016). In addition, p.P397S carriers showed a significantly older age at death (76.3 years vs. 60.3 years; P = 0.009) and longer disease duration (14.0 years vs. 6.9 years; P = 0.002) [fig_ref] Figure 3: Distribution of onset ages, disease durations, and ages at death associated with... [/fig_ref].
# Genetic results
The eight patients studied carried the exon 13 MAPT p.P397S variant. Six of them were probands (subjects I.III.IV, II.II.IX, III.II.VI, III.II.VII, IV.II.V and V.II.III) and the other two (I.III.III and II.II.VIII) were tested after the results found in their relatives. None of them present c9ORF72 expansion. GRN mutations were excluded by direct sequencing in three p.P397S carriers (subjects I.III.IV, II.II.VIII, and IV.II.V) and the rest presented normal serum progranulin levels. In addition, one subject was studied by whole-exome sequencing (II.II.VIII) excluding any other variants in known genes involved in neurodegeneration. This novel variant was predicted as likely pathogenic according to the American College of Medical Genetics and Genomics and the Association for Molecular Pathology guidelines and as definitively pathogenic by Guerreiro et. al. algorithm for mutation's pathogenicity. [bib_ref] Genetic screening of Alzheimer's disease genes in Iberian and African samples yields..., Guerreiro [/bib_ref] [bib_ref] Laboratory Quality Assurance Committee. Standards and guidelines for the interpretation of sequence..., Richards [/bib_ref] In silico analysis produced different results, being predicted pathogenic by SIFT bioinformatics algorithm and probably damaging by Polyphen-2. However, other algorithms scores prediction classified this variant as likely benign (CADD and REVEL algorithms), tolerated (MetaLR), or without a functional consequence (Mutation Assessor). This variant was not described in gnomAD database (http://gnomad.broadinstitute.org/) and it was present at ENSEMBL database as rs1295855402.
# Discussion
In this study, we report a new MAPT variant (p.P397S) in eight subjects from five families suffering from FTLD with autosomal dominant pattern of inheritance. Even if the eight probands belong to five apparently nonrelated families, we could track a common geographical origin in the southeast of Spain, suggesting a possible founder effect of the p.P397S variant in this area.
We hypothesize that this new MAPT variant might be causative of FTLD. According to segregation analysis, their presence in eight affected bvFTD patients and their absence in one unaffected relative is predicted to be likely pathogenic in segregation analysis. In silico analysis produced discrepant results. The p.P397S variant is predicted to destroy a Proline/Serine phosphorylation site at the S396 position. Previous experimental data suggest that phosphorylation at S396 site is necessary to promote the long-term depression at the hippocampus. In this sense, the p.P397S variant potentially will modify the physiological function of tau. [bib_ref] Tau and neurodegenerative disease: the story so far, Iqbal [/bib_ref] [bib_ref] Tau phosphorylation at serine 396 residue is required for hippocampal LTD, Regan [/bib_ref] All patients with the p.P397S variant presented clinical features consistent with the diagnosis of bvFTD. Most patients also developed semantic language impairment. Three patients developed mild rigid-akinetic parkinsonism. These clinical features do not differ from those of patients with the p.P301L mutation. However, patients with the p.P397S MAPT variant showed a significantly older age at disease onset and slower disease progression compared with p.P301L mutation carriers. Neuroimaging of all patients revealed bitemporal atrophy in concordance with the typical atrophy pattern described in FTLD due to MAPT mutations, but with relative preservation of other areas of the brain including frontal areas. [bib_ref] Atrophy patterns in IVS10+16, IVS10+3, N279K, S305N, P301L, and V337M MAPT mutations, Whitwell [/bib_ref] [bib_ref] Distinct patterns of brain atrophy in Genetic Frontotemporal Dementia Initiative (GENFI) cohort..., Fumagalli [/bib_ref] Mutations in the MAPT gene produce FTLD characterized by early onset dementia, with an age of onset between the third and fifth decade of life and 8-10 years of disease duration. [bib_ref] Loss of progranulin function in frontotemporal lobar degeneration, Cruts [/bib_ref] However, previous studies reported that different MAPT mutations may show considerable phenotypic variations. This variability might be explained by the microtubule binding properties of the mutant protein. [bib_ref] Phenotypic variation in hereditary frontotemporal dementia with tau mutations, Van Swieten [/bib_ref] In spite of this, some mutations show an important variability in their age at onset or progression suggesting that other genetic or environmental factors can play an important role in the phenotypic expression. [bib_ref] Variable phenotypic expression and extensive tau pathology in two families with the..., Van Herpen [/bib_ref] Only a few cases of MAPT mutation carriers have been reported to date presenting after the sixth decade. [bib_ref] Autosomal dominant dementia with widespread neurofibrillary tangles, Reed [/bib_ref] [bib_ref] Late-onset frontotemporal dementia with a novel exon 1 (Arg5His) tau gene mutation, Hayashi [/bib_ref] [bib_ref] AnR5Ls mutation in a subject with a progressive supranuclear palsy phenotype, Poorkaj [/bib_ref] Interestingly, other mutations located at the exon 13 are also characterized by a slow rate of disease progression. The p.R406W mutation has a median age of onset of 55 (IQR 51.25-61.75) years and a median disease duration of 14 (IQR 9-26 years). [bib_ref] Slowly progressive dementia caused by MAPT R406W mutations: longitudinal report on a..., Ygland [/bib_ref] As in our series, memory impairment was a marked symptom in p.R406W carriers. The p.T427M MAPT mutation, described only in one family, also seems to have a delayed age at onset (range 60-71 years) and death (67-79 years).However, the p.G389R mutation, also located in exon 13, has been related to an early onset presentation. [bib_ref] Juvenile frontotemporal dementia with parkinsonism associated with tau mutation G389R, Chaunua [/bib_ref] The CSF AD biomarkers were tested in three of the p.P397S carriers (subjects II.II.IX, II.II.VIII, and V.II.III). Surprisingly, levels of p-tau ant t-tau were increased in all three, with decreased levels of Ab42 only in one of them. Several previous reports have described diminished CSF Ab levels in sporadic or/and genetic FTLD. [bib_ref] The wide genetic landscape of clinical frontotemporal dementia: systematic combined sequencing of..., Blauwendraat [/bib_ref] [bib_ref] YKL-40, and neurofilament light in frontotemporal lobar degeneration, Alcolea [/bib_ref] [bib_ref] Ill an-Gala I, et al. Elevated YKL-40 and low sAPPb:YKL-40 ratio in..., Alcolea [/bib_ref] [bib_ref] Cerebrospinal fluid in the differential diagnosis of Alzheimer's disease: clinical utility of..., Paterson [/bib_ref] It is discussed whether this finding is related to an increased deposition of Ab spices or to a reduction of Ab production, as it is associated with a reduction in soluble APP levels. However, in the absence of neuropathological studies in our patients, we cannot rule out the presence of concomitant AD pathology. Of note, the presence of increased p-tau and t-tau at CSF, in addition with the temporal lobe atrophy, some of these patients could be misdiagnosed as suffering from AD. Of course, it is not possible to discard that the presence of AD concomitant pathology was the cause of these results.
The main limitation of this study is the absence of neuropathological and direct functional data. In the absence of functional evidence, we cannot ultimately rule out the possibility that this MAPT variant is just a rare polymorphism. Future functional assays are needed to confirm this variant as a pathogenic mutation.
In conclusion, we report a novel MAPT variant, consisting in the substitution of a Proline for a Serine in the codon 397, in eight subjects from five apparently nonrelated families suffering from frontotemporal dementia with autosomal dominant pattern of inheritance. We hypothesize that this new MAPT variant might be causative of a less aggressive FTLD than other MAPT mutations. In silico analysis using several prediction software produce different results about the potential pathogenicity of this novel variant and segregation analysis predicted it to be likely pathogenic. Thus, in the absence of neuropathology or functional data, we cannot confirm this variant is definitively pathogenic. Future studies are needed in order to determine the pathogenicity of this variant.
[fig] Figure 1: Pedigree of the five reported families. Arrows indicate the probands described in the main text. Upper ages beneath each symbol are age at onset. Lower ages beneath each symbol are age at death. [/fig]
[fig] Figure 2: MRI of subject III.II.VI showing important medial and polar bitemporal atrophy (A). Longitudinal MRI examinations of subject II.II.IX (B). ª 2019 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals, Inc on behalf of American Neurological Association. [/fig]
[fig] Figure 3: Distribution of onset ages, disease durations, and ages at death associated with p.P397S and P301L MAPT carriers (A). Survival curves of p.P397S and P301L MAPT carriers (B). [/fig]
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Non-conducting functions of ion channels: The case of integrin-ion channel complexes
Started as an academic curiosity more than two decades ago, the idea that ion channels can regulate cellular processes in ways that do not depend on their conducting properties (non-ionic functions) gained traction and is now a flourishing area of research. Channels can regulate physiological processes including actin cytoskeletal remodeling, cell motility, excitationcontraction coupling, non-associative learning and embryogenesis, just to mention some, through non-ionic functions. When defective, non-ionic functions can give rise to channelopathies involved in cancer, neurodegenerative disease and brain trauma. Ion channels exert their nonionic functions through a variety of mechanisms that range from physical coupling with other proteins, to possessing enzymatic activity, to assembling with signaling molecules. In this article, we take stock of the field and review recent findings. The concept that emerges, is that one of the most common ways through which channels acquire non-ionic attributes, is by assembling with integrins. These integrin-channel complexes exhibit broad genotypic and phenotypic heterogeneity and reveal a pleiotropic nature, as they appear to be capable of influencing both physiological and pathological processes.ARTICLE HISTORY
## Non-ionic functions of ion channels
Ion channels comprise a fundamental class of integral membrane proteins spanning all three domains of life. Channels make the lipid membrane permeable to ions and for this reason they are present in virtually any cell type. Historically, channels have been considered in light of their conducting properties that range from shaping electrical impulses in excitable cells, to controlling cell volume, secretion, acidification, and other functions in non-excitable cells. However, during the last two decades, we and others, put forward the concept that channels can affect cellular processes in ways that do not depend on their conducting properties (non-ionic functions) [bib_ref] MPS-1 is a K(+) channel beta-subunit and a serine/threonine kinase, Cai [/bib_ref] [bib_ref] TRP-PLIK, a bifunctional protein with kinase and ion channel activities, Runnels [/bib_ref] [bib_ref] Modulation of voltage-dependent Shaker family potassium channels by an aldo-keto reductase, Weng [/bib_ref] [bib_ref] The slowpoke channel binding protein Slob from drosophila melanogaster exhibits regulatable protein..., Zeng [/bib_ref] [bib_ref] Sodium channel β subunits mediate homophilic cell adhesion and recruit ankyrin to..., Malhotra [/bib_ref] [bib_ref] Evidence for a voltage-dependent enhancement of neurotransmitter release mediated via the synaptic..., Mochida [/bib_ref] [bib_ref] Signaling to the nucleus by an L-type calcium channel-calmodulin complex through the..., Dolmetsch [/bib_ref] [bib_ref] Auto-phosphorylation of a voltage-gated K+ channel controls non-associative learning, Cai [/bib_ref]. Much progress has been achieved, and it is now established that channels possess non-ionic functions that they exert through a number of different mechanisms.
In skeletal muscle fibers, voltage-gated L-type calcium channels CACNA1S (Cav1.1) operate as voltage sensors of excitation-contraction (EC) coupling. The CACNA1S channels are physically, and functionally paired, to the ryanodine receptors type 1 (RyR1, [fig_ref] Figure 1: Ion channels exercise non-ionic functions through multiple mechanisms [/fig_ref] ) [bib_ref] Purification of the ryanodine receptor and identity with feet structures of junctional..., Inui [/bib_ref] [bib_ref] Paralysis of frog skeletal muscle fibres by the calcium antagonist D-600, Eisenberg [/bib_ref] [bib_ref] Restoration of excitation-contraction coupling and slow calcium current in dysgenic muscle by..., Tanabe [/bib_ref]. Thus, CACNA1Ss promote sarcoplasmic calcium increase through two distinct mechanisms: by conducting a calcium current-the effective contribution of which to EC is controversial (see ref. [bib_ref] Skeletal muscle CaV1.1 channelopathies, Flucher [/bib_ref] ; and by favoring the release of calcium from the stores through their physical, and thus nonionic, coupling to the RyR1s. Notably, L-type calcium channels and ryanodine receptors are also functionally coupled in the neurons of the brain. The formation of these macromolecular structures is, in turn, helped by delayed rectifier and voltage-gated potassium channel sub-family 2 member 1 (KCNB1, Kv2.1) that acts as scaffolding agent [bib_ref] 1 mediates spatial and functional coupling of L-type calcium channels and ryanodine..., Vierra [/bib_ref] [bib_ref] Remodeling neuronal ER-PM junctions is a conserved nonconducting function of Kv2 plasma..., Kirmiz [/bib_ref] [bib_ref] Induction of stable ER-plasma-membrane junctions by Kv2.1 potassium channels, Fox [/bib_ref] [bib_ref] Kv2 potassium channels form endoplasmic reticulum/plasma membrane junctions via interaction with VAPA..., Johnson [/bib_ref]. KCNB1 is expressed in several neuron types in the brain and in other organs including eyes, pancreas, gastrointestinal tract, kidney, and female reproductive system, where it presumably carries an important repolarizing potassium current [bib_ref] Oxidation of KCNB1 K(+) channels in central nervous system and beyond, Sesti [/bib_ref]. However, KCNB1 exhibits a broad range of non-ionic functions, such as acting as scaffold protein as noted above that will be discussed throughout this review.
Ion channels have a wide repertoire of mechanisms through which to exert non-ionic functions. For example, the transient receptor potential cation channel melastatin-subfamily, member 7 (TRPM7), a non-selective channel, has enzymatic properties [fig_ref] Figure 1: Ion channels exercise non-ionic functions through multiple mechanisms [/fig_ref] [bib_ref] TRP-PLIK, a bifunctional protein with kinase and ion channel activities, Runnels [/bib_ref]. The C-terminus of TRPM7 contains a serine/threonine kinase traceable to the α-kinases family [bib_ref] Identification of a new class of protein kinases represented by eukaryotic elongation..., Ryazanov [/bib_ref] [bib_ref] Characterization of the protein kinase activity of TRPM7/ChaK1, a protein kinase fused..., Ryazanova [/bib_ref]. TRPM7 can phosphorylate several substrates, including proteins involved in actin dynamics, embryogenesis, and neuronal transmission, underscoring the prominent physiological role of TRPM7 [bib_ref] Deletion of Trpm7 disrupts embryonic development and thymopoiesis without altering Mg 2+..., Jin [/bib_ref] [bib_ref] The TRPM7 ion channel functions in cholinergic synaptic vesicles and affects transmitter..., Krapivinsky [/bib_ref] [bib_ref] TRPM7, a novel regulator of actomyosin contractility and cell adhesion, Clark [/bib_ref] [bib_ref] The TRPM7 chanzyme is cleaved to release a chromatin-modifying kinase, Krapivinsky [/bib_ref]. In addition, the α-kinase of TRPM7 indirectly regulates the channel's ionic properties by modulating its sensitivity to magnesium ions and to magnesium nucleotides [bib_ref] TRPM7 channel is regulated by magnesium nucleotides via its kinase domain, Demeuse [/bib_ref].
Functional ion channels are seldom the result of a single-gene product. Typically, they are composed of pore-forming or α-subunits, which assemble together to form the pathway for ions (pore) and thus, a conducting channel, and accessory or βsubunits that modulate the properties of the αsubunits. Several accessory subunits of potassium (K + ) channels, including mammalian Kvβ2, Drosophila melanogaster slowpoke channel-binding protein SLOB, and Caenorhabditis elegans MiRP K + channel accessory Subunit (MPS-1) have enzymatic attributes [bib_ref] MPS-1 is a K(+) channel beta-subunit and a serine/threonine kinase, Cai [/bib_ref] [bib_ref] Modulation of voltage-dependent Shaker family potassium channels by an aldo-keto reductase, Weng [/bib_ref] [bib_ref] The slowpoke channel binding protein Slob from drosophila melanogaster exhibits regulatable protein..., Zeng [/bib_ref]. MPS-1 is an integral membrane protein, homolog to mammalian KCNEs, that has a cytoplasmic domain capable of serine/threonine kinase activity [bib_ref] The MinK-related peptides, Mccrossan [/bib_ref] [bib_ref] A potassium channel-MiRP complex controls neurosensory function in caenorhabditis elegans, Bianchi [/bib_ref]. MPS-1 forms complexes with multiple pore-forming subunits including K + channel Voltage-Sensitive Subunit 1 (KVS-1) and K + channel Habituation to Tap subunit 1 (KHT-1) in the nervous system of the worm [bib_ref] Auto-phosphorylation of a voltage-gated K+ channel controls non-associative learning, Cai [/bib_ref] [bib_ref] A potassium channel-MiRP complex controls neurosensory function in caenorhabditis elegans, Bianchi [/bib_ref]. MPS-1 phosphorylates the α-subunits to decrease their open probability. This mechanism plays an important role in the context of non-associative learning, a universal behavior whereby an organism learns to ignore stimuli that are not important. In the mechanosensory neurons of C. elegans, MPS-1 forms a tripartite complex with KHT-1 and with the acid phosphatase ACP-2, which maintains KHT-1 in a basal, de-phosphorylated state [fig_ref] Figure 1: Ion channels exercise non-ionic functions through multiple mechanisms [/fig_ref] ) [bib_ref] Auto-phosphorylation of a voltage-gated K+ channel controls non-associative learning, Cai [/bib_ref] [bib_ref] An evolutionarily conserved mode of modulation of Shaw -like K + channels, Cotella [/bib_ref]. These tripartite complexes mediate the neurons' response to mechanical stimuli, such as taps to the Petri dish. When the taps are repeated at constant frequency, the animals habituate. The repetitive stimulus triggers the disengagement of ACP-2 from the complex, allowing MPS-1 to phosphorylate KHT-1. This results in a decrease of K + efflux that delays touch-neuron repolarization and, as a consequence, produces temporary desensitization to the mechanical stimuli. Notably, in the neurons of the gigantocellular reticular nucleus, the ACP-2 mammalian homolog, prostatic acid phosphatase (PAP) dephosphorylates KHT-1 homolog, murine KCNC1b, where it may probably underlie adaptation responses [bib_ref] An evolutionarily conserved mode of modulation of Shaw -like K + channels, Cotella [/bib_ref].
Voltage-gated sodium channels (Nav) acquire non-ionic functions through their β-subunits. These proteins not only modulate the ionic properties of the α-subunits; they also mediate cell-to-cell adhesions by acting as molecular linkers that bridge the actin cytoskeleton to neighboring cells. Thus, the β-subunits connect the Nav with the outside, through interacting with proteins such as neurofascins, N-cadherins, and connexins and with the inside, by attaching to the actin cytoskeleton via assembly with ankyrins [fig_ref] Figure 1: Ion channels exercise non-ionic functions through multiple mechanisms [/fig_ref]. The β-subunit promotes direct bonding of Ankyrin G to the Nav α-subunit) [bib_ref] Sodium channel β subunits mediate homophilic cell adhesion and recruit ankyrin to..., Malhotra [/bib_ref] [bib_ref] Heterophilic interactions of sodium channel beta1 subunits with axonal and glial cell..., Mcewen [/bib_ref] [bib_ref] Sodium channel beta1 and beta3 subunits associate with neurofascin through their extracellular..., Ratcliffe [/bib_ref] [bib_ref] Sodium channel beta1 subunit-mediated modulation of Nav1.2 currents and cell surface density..., Mcewen [/bib_ref] [bib_ref] A Common Ankyrin-G-based mechanism retains KCNQ and Na V channels at electrically..., Pan [/bib_ref]. This non-ionic function likely contributes to the distribution of Nav channels in zones where they accumulate at high densities such as the node of Ranvier and the hillock, and to neuritogenesis, as some β-subunits (β1) augment, whereas others (β2) inhibit neurite's outgrowth in cerebellar granule neurons [bib_ref] Sodium channel beta1 subunits promote neurite outgrowth in cerebellar granule neurons, Davis [/bib_ref].
## Integrin-ion channel complexes
A number of ion channels form physical connections with the actin cytoskeleton and with the external environment. However, rather than through their accessory subunits, channels typically achieve those non-ionic functions by interacting with integrins. These are adhesion molecules that connect the extracellular matrix (ECM) to the actin cytoskeleton to regulate the shape, orientation, and movement of cells [bib_ref] Integrin signaling, Giancotti [/bib_ref] [bib_ref] Integrin signaling in cancer: mechanotransduction, stemness, epithelial plasticity, and therapeutic resistance, Cooper [/bib_ref]. In addition, integrins engage intracellular signaling pathways, to control cell proliferation (in the absence of integrin-mediated adhesion and growth factors, cells do not commit to enter the cell cycle, [bib_ref] Integrin-dependent signal transduction regulating cyclin D1 expression and G1 phase cell cycle..., Walker [/bib_ref] , differentiation, survival, and death (anoikis) [bib_ref] Regulation of integrin activation, Kim [/bib_ref].
Integrin signal transduction is complex and extensively interconnected; therefore, a detailed discussion is beyond the scope of this review (for further readings, we direct the reader to refs. [bib_ref] Integrin signaling, Giancotti [/bib_ref] [bib_ref] Integrin signaling in cancer: mechanotransduction, stemness, epithelial plasticity, and therapeutic resistance, Cooper [/bib_ref]. Briefly, in response to anchoragedependent signals, integrins recruit and/or associate with, the integrin adhesome-a cytoskeletal and signaling complex-to control a multitude of cellular functions [bib_ref] The integrin adhesome: from genes and proteins to human disease, Winograd-Katz [/bib_ref] [bib_ref] Definition of a consensus integrin adhesome and its dynamics during adhesion complex..., Horton [/bib_ref] [bib_ref] Functional atlas of the integrin adhesome, Zaidel-Bar [/bib_ref]. Integrins do not possess enzymatic attributes. A fundamental step is the recruitment of Focal Adhesion kinase (FAK), which autophosphorylates Tyr397, creating a binding site for Src kinases. Then, Src, alone or with FAK, phosphorylate several substrates, thereby transducing integrin signals into biochemical events. Integrins promote cell migration by recruiting cytoskeletal linkers, including Talin, and Vinculin, and scaffold/adaptors such as Paxillin and Integrin-linked kinase (ILK) that connect the cytoplasmic tail of integrins to actin filaments. In addition, integrins support cell survival, differentiation, and proliferation, by engaging Ras GTPases, via guanosine-triphosphate exchange factor mSOS. Ras activates several signaling pathways including Mitogen Activated Protein kinase (MAPK) cascades and Phosphoinositide 3 kinase-Protein kinase B-mammalian target of rapamycin (PI3K-Akt-mTOR) signaling that lead to the phosphorylation of cytoplasmic targets and to the enhancement or repression of nuclear transcription.
Integrin-channel relationships play prominent roles in disease, primarily cancer, given that several aspects of these pathologies, for instance, tumor invasion, differentiation, and metastasis involve cellular functions that require the coordinated action of integrins . In addition, the expression of channels or integrins may adjust the expression of one another to maintain cellular homeostasis. In these cases, increased integrin expression may be associated with channel's recruitment to the plasma membrane and/or boosted channel activity and vice versa. It is also possible that in extreme and/or pathological conditions the increased presence of one may compensate for the absence of the other [bib_ref] Voltage-Gated K+ channel, Kv3.3 is involved in hemin-induced K562 differentiation, Song [/bib_ref] [bib_ref] Crosstalk between CD11b and Piezo1 mediates macrophage responses to mechanical cues, Atcha [/bib_ref] [bib_ref] The voltagegated K(+) channel Kv1.3 modulates platelet motility and α(2)β(1) integrin-dependent adhesion..., Wright [/bib_ref]. The first example of cooperation between ion channels and integrins comes from L-type calcium channels expressed in smooth muscle cells of small blood vessels. In rat arterioles, integrin signaling favors vasoconstriction by potentiating CACNA1C (Cav1.2) channels [bib_ref] Modulation of calcium current in arteriolar smooth muscle by alphav beta3 and..., Wu [/bib_ref] [bib_ref] Regulation of the L-type calcium channel by alpha 5 beta 1 integrin..., Wu [/bib_ref] [bib_ref] β 1 integrin activation of L-type calcium channels in vascular smooth muscle..., Waitkus-Edwards [/bib_ref]. Integrin ligands induce the assembly of integrins α 5 β 1 with CACNA1C channels to form macromolecular complexes with protein kinase A (PKA), and c-Src tyrosine kinases [bib_ref] Spatial association of the Cav1.2 calcium channel with α 5 β 1..., Chao [/bib_ref]. Following integrin engagement, c-Src phosphorylates CACNA1C leading to current potentiation that facilitates vasoconstriction [bib_ref] Integrin receptor activation triggers converging regulation of Cav1.2 calcium channels by c-Src..., Gui [/bib_ref]. Interestingly, α 5 β 1 integrins modulate large conductance, calcium-activated K + (BK) channels in the same vessels, through largely similar mechanisms [bib_ref] Alpha5beta1 integrin engagement increases large conductance, Ca2+--activated K+ channel current and Ca2+..., Yang [/bib_ref] [bib_ref] Potentiation of large conductance, Ca 2+ -activated K + (BK) channels by..., Wu [/bib_ref]. The mechanosensitive ion channel PIEZO1 provides an example of a channel regulating integrin signaling [bib_ref] Piezo1 and Piezo2 are essential components of distinct mechanically activated cation channels, Coste [/bib_ref]. PIEZO1 is overexpressed in aggressive cancers at focal adhesions where it augments tissue stiffening and tumor cell proliferation [bib_ref] Identification of PIEZO1 as a potential prognostic marker in gliomas, Zhou [/bib_ref] [bib_ref] Adherent cell remodeling on micropatterns is modulated by Piezo1 channels, Jetta [/bib_ref]. An increase in PIEZO1 current causes the activation of integrin-FAK signaling that reinforces tissue hardening. In turn, the firmer mechanical microenvironment boosts PIEZO1 expression and promotes tumor cell proliferation in a sort of auto-catalytic process [bib_ref] A feedforward mechanism mediated by mechanosensitive ion channel PIEZO1 and tissue mechanics..., Chen [/bib_ref]. Another example of a channel able to modulate integrin function is chloride intracellular channel, CLIC1. This protein promotes integrin-mediated, cell-matrix adhesion and the signaling for cytoskeleton . Non-ionic functions of integrin channel complexes (a) A widespread mechanism by which channels acquire nonionic functions is by forming macromolecular complexes with integrins. Generally, these integrin-channel complexes regulate the shape, orientation, and movement of cells through the integrin machinery. In addition, they modulate cell proliferation, differentiation, survival and death, and are therefore implicated in a variety of oncogenic processes of different etiologies. (b) Integrin-α5-KCNB1 complexes operate in the neurons of the brain (for simplicity the ECM is not depicted). Under conditions of oxidative stress, the KCNB1 channels form oligomers that trigger apoptosis. The molecular steps underlying this process include the engagement of FAK and Src by the integrins, followed by the activation of a canonical Ras-MAPK cascade. Killer kinases, such as JNK and p38 and caspases execute the apoptotic program. At the same time, these IKCs neutralize a major mechanism of cell survival by sequestering Akt, that cannot be activated (phosphorylated) and released into the cytoplasm, to phosphorylate its multiple substrates including BAD. extension during tumor cell migration and invasion [bib_ref] CLIC1 recruits PIP5K1A/C to induce cell-matrix adhesions for tumor metastasis, Peng [/bib_ref]. Integrins signal through FAK and the critical role of this protein kinase in promoting cell motility is well established [bib_ref] TRPM4 Is a novel component of the adhesome required for focal adhesion..., Cáceres [/bib_ref] [bib_ref] Formation of Kv2.1-FAK complex as a mechanism of FAK activation, cell polarization..., Wei [/bib_ref]. In some cancer types, however, FAK activity is decreased and with that, cellular proliferation and invasion. This inhibition of FAK activity is mediated by the formation of tripartite FAK, integrin-β4, and calcium-activated chloride channel protein (mCLCA1) complexes. Thus, overexpression of the same leads to phosphorylation and inhibition of FAK and ERK proteins [bib_ref] Along with its favorable prognostic role, CLCA2 inhibits growth and metastasis of..., Qiang [/bib_ref].
## Integrin-k + channel complexes are widely expressed
Several macromolecular complexes formed by integrins and K + channels, generically named Integrin-K + channel complexes or IKCs, have been identified and characterized [bib_ref] Voltage-Gated K+ channel, Kv3.3 is involved in hemin-induced K562 differentiation, Song [/bib_ref] [bib_ref] The voltagegated K(+) channel Kv1.3 modulates platelet motility and α(2)β(1) integrin-dependent adhesion..., Wright [/bib_ref] [bib_ref] Alpha5beta1 integrin engagement increases large conductance, Ca2+--activated K+ channel current and Ca2+..., Yang [/bib_ref] [bib_ref] Formation of Kv2.1-FAK complex as a mechanism of FAK activation, cell polarization..., Wei [/bib_ref] [bib_ref] Altering integrin engagement regulates membrane localization of K(ir)2.1 channels, Sengupta [/bib_ref] [bib_ref] β1-Integrin-and KV1.3 channel-dependent signaling stimulates glutamate release from Th17 cells, Birkner [/bib_ref] [bib_ref] Complementary roles of KCa3.1 channels and β1-integrin during alveolar epithelial repair, Girault [/bib_ref] [bib_ref] Oxidation of KCNB1 potassium channels triggers apoptotic integrin signaling in the brain, Yu [/bib_ref] [bib_ref] Complexes formed with integrin-α5 and KCNB1 potassium channel wild type or epilepsy-susceptibility..., Yu [/bib_ref] [bib_ref] Oxidation of KCNB1 channels in the human brain and in mouse model..., Wei [/bib_ref] [bib_ref] Antagonistic roles of Ras-MAPK and Akt signaling in integrin-K + channel complex-mediated..., Forzisi [/bib_ref] [bib_ref] An inward rectifier K+ current modulates in neuroblastoma cells the tyrosine phosphorylation..., Bianchi [/bib_ref] [bib_ref] Integrinmediated neurite outgrowth in neuroblastoma cells depends on the activation of potassium..., Arcangeli [/bib_ref] [bib_ref] Response to fibronectin-integrin interaction in leukaemia cells: delayed enhancing of a K+..., Becchetti [/bib_ref] [bib_ref] HERG K + channels and β1 integrins interact through the assembly of..., Cherubini [/bib_ref] [bib_ref] Human ethera-go-go -related Gene 1 channels are physically linked to β 1..., Cherubini [/bib_ref] [bib_ref] HERG K+ channels activation during beta(1) integrin-mediated adhesion to fibronectin induces an..., Hofmann [/bib_ref] [bib_ref] Harnessing the hERG1/β1 integrin complex via a novel bispecific single-chain antibody: an..., Duranti [/bib_ref] [bib_ref] Extracellular K (+) and opening of voltage-gated potassium channels activate T cell..., Levite [/bib_ref]. Arcangeli and colleagues were the first to reveal the existence of pathophysiological links between integrins and K + channels, when they showed that murine erythroleukemia cell adhesion to fibronectin, and neurite outgrowth of neuroblastoma cells were associated with a potassium current, later attributed to the voltage-gated K + channel KCNH2 (synonyms, Kv11.1, HERG, and hERG1) [bib_ref] Integrinmediated neurite outgrowth in neuroblastoma cells depends on the activation of potassium..., Arcangeli [/bib_ref] [bib_ref] A novel inward-rectifying K+ current with a cell-cycle dependence governs the resting..., Arcangeli [/bib_ref] [bib_ref] Soluble or bound laminin elicit in human neuroblastoma cells short-or long-term potentiation..., Arcangeli [/bib_ref] [bib_ref] herg encodes a K+ current highly conserved in tumors of different histogenesis:..., Bianchi [/bib_ref] [bib_ref] Long-term exposure to retinoic acid induces the expression of IRK1 channels in..., Arcangeli [/bib_ref] [bib_ref] Modulation of HERG current and herg gene expression during retinoic acid treatment..., Arcangeli [/bib_ref]. Those initial findings were followed by a series of seminal studies that demonstrated that integrin-mediated cell adhesion of KCNH2 promotes cell differentiation. Most importantly, the activation of KCNH2 channels proceeds through integrin-β1 which was demonstrated to physically interact with the channel [bib_ref] Human ethera-go-go -related Gene 1 channels are physically linked to β 1..., Cherubini [/bib_ref]. Overall, that body of work shows that integrin-β1-KCNH2 complexes integrate the signaling evoked by cell adhesion to the ECM, with the cell differentiation machinery. The pathological implications of integrin-β1-KCNH2 complexes are significant, when one considers that cell differentiation can play a role in oncogenesis. Accordingly, KCNH2 is highly conserved in tumors of different histogenesis, and integrin-β1-KCNH2 complexes play a central role in cancer formation and progression [bib_ref] Harnessing the hERG1/β1 integrin complex via a novel bispecific single-chain antibody: an..., Duranti [/bib_ref] [bib_ref] A novel inward-rectifying K+ current with a cell-cycle dependence governs the resting..., Arcangeli [/bib_ref] [bib_ref] herg encodes a K+ current highly conserved in tumors of different histogenesis:..., Bianchi [/bib_ref] [bib_ref] HERG potassium channels are constitutively expressed in primary human acute myeloid leukemias..., Pillozzi [/bib_ref] [bib_ref] hERG1 channels modulate integrin signaling to trigger angiogenesis and tumor progression in..., Crociani [/bib_ref]. Furthermore, in colorectal cancer cell lines, the binding of fibronectin and collagen I to integrin-β1 at the ECM level promotes the formation of a tripartite complex composed, other than of integrin-β1, of KCNH2 and of the Na + /H + antiporter NHE1. The activity of this tripartite complex regulates the cytosolic pH of colorectal cancer cells, thereby contributing to the maintenance of tumor microenvironment [bib_ref] K(V)11.1 potassium channel and the Na(+)/H(+) Antiporter NHE1 modulate adhesion-dependent intracellular pH..., Iorio [/bib_ref].
The voltage-gated K + channel KCNB1 forms stable complexes with integrin-α5 in neurons of the brain [bib_ref] Oxidation of KCNB1 potassium channels triggers apoptotic integrin signaling in the brain, Yu [/bib_ref] [bib_ref] Oxidation of KCNB1 channels in the human brain and in mouse model..., Wei [/bib_ref]. It is also likely that these complexes exist in other tissues, including the retina and the pancreas, where both KCNB1 and integrin-α5 are present. Studies carried out in heterologous expression systems indicated that the activity of KCNB1 is translated by the integrins into biochemical events-mediated by FAK, Src, Ras GTPases, MAPKs, and protein kinase Akt-to advance the development of actin-rich cellular protrusions in Chinese hamster ovary (CHO) cells, stimulating their motility and to enhance neuritogenesis of neuroblastoma cells [bib_ref] Formation of Kv2.1-FAK complex as a mechanism of FAK activation, cell polarization..., Wei [/bib_ref] [bib_ref] Oxidation of KCNB1 potassium channels triggers apoptotic integrin signaling in the brain, Yu [/bib_ref] [bib_ref] Complexes formed with integrin-α5 and KCNB1 potassium channel wild type or epilepsy-susceptibility..., Yu [/bib_ref] [bib_ref] Oxidation of KCNB1 channels in the human brain and in mouse model..., Wei [/bib_ref] [bib_ref] Antagonistic roles of Ras-MAPK and Akt signaling in integrin-K + channel complex-mediated..., Forzisi [/bib_ref] [bib_ref] Molecular mechanisms underlying the apoptotic effect of KCNB1 K+ channel oxidation, Wu [/bib_ref] [bib_ref] Hypoxic preconditioning enhances bone marrow mesenchymal stem cell migration via Kv2.1 channel..., Hu [/bib_ref]. Accordingly, Srcmediated phosphorylation of KCNB1 at Tyr124 is critical for the proliferation and myelination of murine Schwann cells [bib_ref] Constitutive activation of delayed-rectifier potassium channels by a src family tyrosine kinase..., Sobko [/bib_ref] [bib_ref] Tyrosine kinases modulate K + channel gating in mouse Schwann cells, Peretz [/bib_ref]. These functions are predominantly non-ionic in nature. Indeed, while certain non-conducting KCNB1 variants implicated in severe epileptic syndromes engage integrin signaling and stimulate cell migration, other (non-conducting) variants that fail to activate the same cascades do not enhance cell migration [bib_ref] Complexes formed with integrin-α5 and KCNB1 potassium channel wild type or epilepsy-susceptibility..., Yu [/bib_ref]. Notably, all those variants are associated with large phenotypic heterogeneity [bib_ref] Neurodevelopmental disorders caused by de novo variants in KCNB1 genotypes and phenotypes, De Kovel [/bib_ref]. Overall, it appears that integrin-α5-KCNB1 complexes translate membrane excitability into intracellular signals important for cellular plasticity. This implies that when defective, integrin-α5-KCNB1 complexes could cause neurological disease through a variety of mechanisms ranging from impaired conduction to dysregulated integrin signaling. In fact, the non-ionic, pleiotropic, nature of integrin-α5-KCNB1 complexes becomes relevant in neurodegenerative diseases, when neurons are subject to stressful conditions, namely oxidative stress.
## Integrin-α5-kcnb1 complexes trigger programmed cell death
The non-ionic functions of KCNB1 began to unravel after it was discovered that KCNB1 is a pro-apoptotic protein. Aizenman and collaborators showed that cells expressing KCNB1 undergo apoptosis when challenged by oxidants [bib_ref] Mediation of neuronal apoptosis by Kv2.1-encoded potassium channels, Pal [/bib_ref]. Subsequent studies revealed that oxidative stress (an imbalance between the oxidants present in the cell and its antioxidant defenses) correlated well with the activation of a number of protein kinases, including p38 MAPK and Src family of tyrosine kinases that phosphorylated KCNB1 at Ser800 and Tyr124 (the latter residue is also responsible for Srcmediated increase of myelination and proliferation of mouse Schwann cells) [bib_ref] Constitutive activation of delayed-rectifier potassium channels by a src family tyrosine kinase..., Sobko [/bib_ref] [bib_ref] Tyrosine kinases modulate K + channel gating in mouse Schwann cells, Peretz [/bib_ref] [bib_ref] Obligatory role of ASK1 in the apoptotic surge of K+ currents, Aras [/bib_ref] [bib_ref] Apoptotic surge of potassium currents is mediated by p38 phosphorylation of Kv2, Redman [/bib_ref] [bib_ref] Regulation of apoptotic potassium currents by coordinated zinc-dependent signalling, Redman [/bib_ref] [bib_ref] Phosphorylationdependent regulation of Kv2.1 channel activity at tyrosine 124 by Src and..., Tiran [/bib_ref]. The phosphorylation of the channel enhances its interactions with syntaxin accelerating insertion into the plasma membrane [bib_ref] Convergent Ca 2+ and Zn 2+ signaling regulates apoptotic Kv2.1 K +..., Mccord [/bib_ref]. The increased K + efflux that follows, stimulates caspase and nuclease activity and marks a point of no-return toward apoptosis. However, we later discovered that reactive oxygen species (ROS) can directly modify KCNB1 proteins, turning them into aberrant, toxic channels [bib_ref] Toxic role of K+ channel oxidation in mammalian brain, Cotella [/bib_ref]. The journey into what at the time was uncharted territory began when we tested the idea that the excess ROS that build up in aging cells may oxidize K + channels, leading to neuronal failure. For that exploratory inquiry, we took advantage of the simplicity of C. elegans, which indeed turned out to be an excellent tool to capture the essence of the problem. Accordingly, the KVS-1 channel, which is a homolog of KCNB1, becomes progressively oxidated at Cys113 in the sensory neurons of aging worms [bib_ref] A potassium channel-MiRP complex controls neurosensory function in caenorhabditis elegans, Bianchi [/bib_ref] [bib_ref] Oxidation of a potassium channel causes progressive sensory function loss during aging, Cai [/bib_ref]. The oxidative modifications alter the gating of KVS-1, which by impairing sensory neuron excitability, leads to behavioral deficit. The cysteine responsible for the functional alterations of the KVS-1 channel is conserved in KCNB1 (Cys73. The KVS-1 channel possesses a 40 amino-acid domain composed of a N-inactivating ball preceded by an N inactivation regulatory domain, called NIRD which modulates the inactivation of the channel [bib_ref] A new mode of regulation of N-type inactivation in a Caenorhabditis elegans..., Cai [/bib_ref]. This suggests that also KCNB1 may be susceptible to redox. In fact, oxidants cross-link KCNB1 subunits to each other (oligomers), by inducing the formation of disulfide bridges involving conserved Cys73 [bib_ref] Toxic role of K+ channel oxidation in mammalian brain, Cotella [/bib_ref]. Most importantly, KCNB1 oligomers were detected in the post mortem hippocampi of male and female AD donors (83.8 ± 0.79 yrs. average age) where they were significantly more abundant than in age-matched controls (82.5 ± 0.76 yrs.) [bib_ref] Oxidation of KCNB1 channels in the human brain and in mouse model..., Wei [/bib_ref]. Just to give an idea of the extent of KCNB1 oligomerization in the aging human brain, ~40% of KCNB1 channels were found to be oligomerized in control donors, and this number increased to ~75% in Alzheimer's donors. Similarly, KCNB1 oligomers were found to be ~35% and ~80% in, respectively, 22 month-old control and 3xTg-AD mice [bib_ref] Toxic role of K+ channel oxidation in mammalian brain, Cotella [/bib_ref]. KCNB1 oligomers do not conduct current [bib_ref] Toxic role of K+ channel oxidation in mammalian brain, Cotella [/bib_ref]. Studies in 3xTg-AD mouse model of Alzheimer's disease-a pathology characterized by extensive oxidative stress-showed that oxidized KCNB1 channels impair neuron repolarization causing hippocampal hyperexcitability [bib_ref] Altered Kv2.1 functioning promotes increased excitability in hippocampal neurons of an Alzheimer's..., Frazzini [/bib_ref] [bib_ref] Tripletransgenic model of Alzheimer's disease with plaques and tangles: intracellular Abeta and..., Oddo [/bib_ref]. However, the toxicity of KCNB1 oligomers does not stem only from their lack of conduction, and is also caused by nonionic mechanisms. A KCNB1 variant obtained by replacing Cys73 to Ala (C73A) does not form oligomers and conducts normally. Therefore, C73A channels should give rise to an apoptotic current surge in response to an oxidative insult. In contrast, the mutant does not cause cellular death. This implies that the formation of oligomers, rather than KCNB1 current, is the event that triggers the initial pro-apoptotic stimulus.
## Integrin-α5-kcnb1 complexes are implicated in multiple pathologies
Alzheimer's disease and Traumatic Brain Injury (TBI) provide two well characterized examples of the pleiotropic nature of integrin-α5-KCNB1 complexes. As the brain undergoes degeneration or trauma, these IKCs turn pathogenic by promoting inflammation and apoptosis via integrins and their signaling machinery [bib_ref] Oxidation of KCNB1 channels in the human brain and in mouse model..., Wei [/bib_ref] [bib_ref] Antagonistic roles of Ras-MAPK and Akt signaling in integrin-K + channel complex-mediated..., Forzisi [/bib_ref] [bib_ref] Oxidation of KCNB1 potassium channels causes neurotoxicity and cognitive impairment in a..., Yu [/bib_ref]. The signaling pathways recruited by integrin-α5-KCNB1 complexes have been characterized in detail. Conformational changes in KCNB1 such as opening and closing leads to the recruitment of FAK. The kinase autophosphorylates Tyr397, creating a binding site for Src family tyrosine kinases. FAK/Src complexes activate small GTPases of the Ras sub-family, which in turn, set in motion a canonical MAPK pathway, composed of Rapidly Activated fibrosarcoma (RAF) kinase, Mitogen-Activated Protein Kinase Kinase (MEK) and Extracellular signal-Regulated kinase (ERK) [bib_ref] Oxidation of KCNB1 potassium channels triggers apoptotic integrin signaling in the brain, Yu [/bib_ref] [bib_ref] Antagonistic roles of Ras-MAPK and Akt signaling in integrin-K + channel complex-mediated..., Forzisi [/bib_ref]. This is followed by the appearance of killer kinases, including C-Jun N-Terminal Kinase (JNK) and presumably kinases implicated in the surge mechanism such as p38 MAPK, caspases and other death proteins, that execute the final steps of the apoptotic program [bib_ref] Molecular mechanisms underlying the apoptotic effect of KCNB1 K+ channel oxidation, Wu [/bib_ref] [bib_ref] Apoptotic surge of potassium currents is mediated by p38 phosphorylation of Kv2, Redman [/bib_ref] [bib_ref] Oxidation of KCNB1 potassium channels causes neurotoxicity and cognitive impairment in a..., Yu [/bib_ref].
## Integrin-α5-kcnb1 complexes keep balance between cell's life and death
Studies of integrin-α5-KCNB1 complexes expressed in immortal cells demonstrated that the activation of Ras-MAPK signaling represents a causative step toward apoptosis [bib_ref] Complexes formed with integrin-α5 and KCNB1 potassium channel wild type or epilepsy-susceptibility..., Yu [/bib_ref] [bib_ref] Antagonistic roles of Ras-MAPK and Akt signaling in integrin-K + channel complex-mediated..., Forzisi [/bib_ref]. However, also IKCs formed with anti-apoptotic C73A subunits turned out to engage the same Ras-MAPK cascades. The answer to this conundrum is that the difference between IKCs formed with WT and C73A KCNB1 channels resides in the way they regulate active Akt, a major architect of cell survival. Akt keeps apoptosis progression in check through phosphorylating BCL2 associated agonist of cell death (BAD) at Ser136 [bib_ref] Survival signaling goes BAD, Bergmann [/bib_ref] [bib_ref] Regulation of apoptosis by Bcl-2 family proteins, Burlacu [/bib_ref]. When BAD is dephosphorylated, it forms heterodimers with Bcl-2 and Bcl-xL, preventing them from inhibiting the release of cytochrome c through the mitochondrial pore [bib_ref] Regulation of apoptosis by Bcl-2 family proteins, Burlacu [/bib_ref]. Accordingly, Akt is significantly more active, and with it, BAD is more phosphorylated, in the presence of IKCs formed with C73A mutants compared to WT [bib_ref] Complexes formed with integrin-α5 and KCNB1 potassium channel wild type or epilepsy-susceptibility..., Yu [/bib_ref] [bib_ref] Antagonistic roles of Ras-MAPK and Akt signaling in integrin-K + channel complex-mediated..., Forzisi [/bib_ref]. Furthermore, pharmacological inhibition of Akt abolishes the protective effect of C73A, but when Ras-MAPK signaling is simultaneously inhibited, apoptosis is also suppressed. The activation of Akt occurs at the plasma membrane, where the kinase is sequentially phosphorylated at Thr308 and at Ser473 before being released back into the cytoplasm [bib_ref] PI3K-PKB/Akt pathway, Hemmings [/bib_ref]. An oxidative insult causes selective increase of Akt binding to WT channels that prevents the kinase from being phosphorylated and released into the cytoplasm [bib_ref] Antagonistic roles of Ras-MAPK and Akt signaling in integrin-K + channel complex-mediated..., Forzisi [/bib_ref]. At the moment, the causes for the selective affinity of oxidized WT channels for Akt are not known. An important fact to consider, is that KCNB1 oligomers are poorly endocytosed and consequently build up in the plasma membrane [bib_ref] Molecular mechanisms underlying the apoptotic effect of KCNB1 K+ channel oxidation, Wu [/bib_ref]. Hence, it is possible that the increased presence of KCNB1 protein at the membrane might enhance the probability of interacting with Akt. Indeed, under normal conditions, a small fraction of KCNB1 channels, either WT and C73A, co-immunoprecipitate with Akt [bib_ref] Antagonistic roles of Ras-MAPK and Akt signaling in integrin-K + channel complex-mediated..., Forzisi [/bib_ref]. In summary, the evidence at hand provides a model for the toxicity of integrin-α5-KCNB1 complexes that predicts that these IKCs send apoptotic stimuli via Ras-MAPK cascades, while simultaneously neutralizing the mechanisms of cellular survival.
## Biomedical relevance of integrin-α5-kcnb1 complexes
The elucidation of the non-ionic functions of IKCs carries important biomedical implications, as drugs that impinge on the signaling pathways engaged by these complexes have the potential to ameliorate multiple pathologies. One promising candidate is Dasatinib, a second-generation Src tyrosine kinase inhibitor. Dasatinib is FDAapproved for the treatment of Philadelphia chromosome-positive (Ph+) chronic myeloid leukemia (CML) and acute lymphoblastic leukemia including Central Nervous System CMS (the drug is blood-brain barrier permeable) [bib_ref] Remission of Philadelphia chromosome-positive central nervous system leukemia after dasatinib therapy, Abdelhalim [/bib_ref] [bib_ref] Dasatinib crosses the blood-brain barrier and is an efficient therapy for central..., Porkka [/bib_ref] [bib_ref] Dasatinib in the management of lymphoid blast crisis of Philadelphia-positive chronic myeloid..., Alimena [/bib_ref] [bib_ref] Excellent response to dasatinib of childhood Philadelphia positive intracranial acute lymphoblastic leukaemia..., Bhadri [/bib_ref] [bib_ref] Dasatinib induces complete remission in a patient with primary cerebral involvement of..., Russwurm [/bib_ref] [bib_ref] Isolated central nervous system relapse in patient with blast-crisis chronic myeloid leukemia..., Zhou [/bib_ref] [bib_ref] Dasatinib maintenance therapy after allogeneic hematopoietic stem cell transplantation for an isolated..., Nishimoto [/bib_ref] [bib_ref] The overwhelmingly positive response to Dasatinib of a patient with multiple blast..., Xu [/bib_ref] [bib_ref] Dasatinib as the salvage therapy for chronic myeloid leukemia with blast crisis..., Lai [/bib_ref]. Dasatinib, reverses cognitive decline in rodent models of AD by decreasing β-amyloid (Aβ) load and neurofibrillary tau tangles (NFT), inflammation, and oxidative stress [bib_ref] Oxidation of KCNB1 channels in the human brain and in mouse model..., Wei [/bib_ref] [bib_ref] Tau protein aggregation is associated with cellular senescence in the brain, Musi [/bib_ref] [bib_ref] Inhibition of Src kinase activity attenuates amyloid associated microgliosis in a murine..., Dhawan [/bib_ref] [bib_ref] Amyloid-beta oligomers stimulate microglia through a tyrosine kinase dependent mechanism, Dhawan [/bib_ref] [bib_ref] Senolytic therapy alleviates Abeta-associated oligodendrocyte progenitor cell senescence and cognitive deficits in..., Zhang [/bib_ref]. The potential therapeutic effects of Dasatinib in AD stem from its ability to impinge on multiple cellular mechanisms, which share the involvement of Src tyrosine kinases, such as the oxidation of integrin-α5-KCNB1 complexes [bib_ref] Oxidation of KCNB1 channels in the human brain and in mouse model..., Wei [/bib_ref] [bib_ref] Oxidation of KCNB1 potassium channels causes neurotoxicity and cognitive impairment in a..., Yu [/bib_ref]. Combs and colleagues were the first to show that the inhibition of Src tyrosine kinases by Dasatinib acted to reduce brain inflammation and improved cognitive outcome in mouse model of Alzheimer's disease [bib_ref] Inhibition of Src kinase activity attenuates amyloid associated microgliosis in a murine..., Dhawan [/bib_ref] [bib_ref] Amyloid-beta oligomers stimulate microglia through a tyrosine kinase dependent mechanism, Dhawan [/bib_ref]. Work from our lab further indicated that prolonged Dasatinib treatment in 3xTg-AD mice decreased brain inflammation and Aβ load and reduced behavioral deficit caused by the oxidation of integrin-α5-KCNB1 complexes [bib_ref] Oxidation of KCNB1 channels in the human brain and in mouse model..., Wei [/bib_ref]. Dasatinib was also found to significantly decrease inflammation and neurodegeneration caused by oxidation of integrin-α5-KCNB1 complexes in the Lateral Fluid Percussion (LFP) mouse model of brain trauma, a condition that shares with Alzheimer's disease copious oxidative stress and Aβ plaque formation [bib_ref] A beta 42 is the predominant form of amyloid beta-protein in the..., Gentleman [/bib_ref] [bib_ref] beta A4 amyloid protein deposition in brain after head trauma, Roberts [/bib_ref] [bib_ref] Beta amyloid protein deposition in the brain after severe head injury: implications..., Roberts [/bib_ref] [bib_ref] Distribution of beta-amyloid protein in the brain following severe head injury, Graham [/bib_ref] [bib_ref] Alzheimer's pathology in human temporal cortex surgically excised after severe brain injury, Ikonomovic [/bib_ref] [bib_ref] Oxidative stress in traumatic brain injury, Rodríguez-Rodríguez [/bib_ref]. Orr and colleagues detected a reduction in total NFT density, neuron loss, and ventricular enlargement following Dasatinib+Quercetin (a flavonoid found in many plants and foods) regimen in human Alzheimer's neurons and in the brains of a mouse model of tauopathy [bib_ref] Tau protein aggregation is associated with cellular senescence in the brain, Musi [/bib_ref]. Others have sought to identify agents targeting the surge mechanism, which relies on the interaction between the C-terminus of KCNB1 and syntaxin. This effort has led to the identification of a small molecule inhibitor (cpd5) of the protein-protein interaction between syntaxin and KCNB1, that has shown some efficacy in ameliorating neuronal loss in middle cerebral artery occlusion mouse model of ischemic stroke [bib_ref] Targeting a potassium channel/syntaxin interaction ameliorates cell death in ischemic stroke, Yeh [/bib_ref]. Currently, seven drugs that inhibit Ras-MAPK signaling are FDA-approved for the treatment of multiple cancer pathologies, and Akt agonists are being developed [bib_ref] Current development status of MEK inhibitors, Cheng [/bib_ref] [bib_ref] New perspectives for targeting RAF kinase in human cancer, Karoulia [/bib_ref] [bib_ref] LaSota strain expressing the rabies virus glycoprotein (rL-RVG) suppresses gastric cancer by..., Bu [/bib_ref] [bib_ref] Drugging the undruggable: advances on RAS targeting in cancer, Molina-Arcas [/bib_ref]. It is therefore to be hoped that in the future, some of the drugs that target components of the integrin-α5-KCNB1 complexes signaling machinery could be repurposed for the treatment of diseases, including Alzheimer's disease, TBI, and stroke.
# Conclusions
In a short period of time, enormous progress has been achieved in our understanding of ion channels and their non-ionic functions. In 20 years, what started as sporadic, anecdotal evidence has become a solid, broad field of research. Channels exert their non-ionic functions through various mechanisms, ranging from physical coupling, to possessing enzymatic features, and many other mechanisms will likely be discovered as our understanding of these proteins progresses. The repertoire of non-ionic functions of ion channels is implicated in a broad range of physiological processes, as fundamental as actin cytoskeleton remodeling and cell migration, differentiation, embryogenesis, excitation-contraction coupling, and learning and memory formation. Consequently, defective non-ionic functions give rise to pathologies, including TBI, Alzheimer's disease, stroke, and cancer.
Interestingly, channels achieve their non-ionic functions by primarily interacting with integrins. The voltage-gated and delayed rectifier K + channel KCNB1 provides one of the best examples of the broad pathophysiological implications of nonionic functions of an ion channel. Integrin-α5-KCNB1 complexes are involved in regulating basic cellular processes, and they become toxic in pathological conditions. The elucidation of these mechanisms may provide pharmacological indications that could be quickly translated to human clinical trials.
[fig] Figure 1: Ion channels exercise non-ionic functions through multiple mechanisms (a) Channels perform non-ionic functions through physical coupling with other channels, or posses enzymatic domains in their α-subunits (b), or β-subunits (c). In addition to playing canonical regulatory roles, the β-subunits of Nav channels act as adhesion molecules that help forming cell-to-cell contacts, or link the actin cytoskeleton to the extracellular matrix, (d). [/fig]
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Gene Environment Interactions in the Etiology of Neural Tube Defects
## Neural tube development
The developing nervous system in vertebrates arises initially from the neural plate, an embryonic layer of cells that is specified and differentiates from the ectoderm immediately after gastrulation proceeds. The layer of cells located at the central portion of the neural plate will give rise to the central nervous system, while on its periphery, adjacent to the neural groove, neural crest cells will be transiently formed before delamination and migration to the adjacent paraxial mesoderm. The next step is neurulation, a morphogenetic process in which the neural plate will bend over itself forming the neural tube. The most anterior portion of this developing tube will form the cephalic structures of the central nervous system, while the spinal cord will be formed by the truncal and caudal portions located posteriorly on the tube. In a highly regulated manner following neurulation, the neural crest will migrate ventrally to colonize specific sites prior to differentiating into a range of diverse cell types.
In vertebrate embryos, the adult brain originates from the most rostral anterior portion of the developing neural tube, while the spinal cord originates from the most posterior caudal extremity. Failure during neural tube closure can occur in different regions during the developmental axis formation, resulting in different abnormalities. Anencephaly cases occur when the anterior section of the developing neural tube fails to properly fuse, resulting in incomplete development of both the skull and the brain. Failure of neural tube fusion in the most caudal portions of the embryo results in the malformation commonly referred to as spina bifida. This malformation is more common, and distinctions are made between closed (occulta) and open (aperta) defects. Spina bifida aperta involves exteriorized neural tissue that is not fully covered by skin. When a cystic protrusion is detected, there the two primary subtypes referred to as meningocele, when the protrusion involves the meninges along with cerebrospinal fluid, and myelomeningocele, when the cystic protrusion contains the portions of the developing spinal cord. Finally, closed spina bifida occulta is when the defect is covered by skin. Cases, where spina bifida occulta occurs, can evolve asymptomatically throughout life and might never be detected.
When the embryonic neural tube closure reaches its final stage, it is possible to identify the neuroepithelium, which is composed by a monolayer of bipolar cells, delimiting the ventricular lumen. The basal region of the neuroepithelium contacts the basal lamina at the periphery of the tube, while the apical region is in contact with the lumen or ventricle. Neurons are generated only by the lateral regions of the neural tube, with the roof and floor plates responsible for the production of morphogens that standardize the different neural types along the dorsoventral axis. Within the primordial spinal cord, the neural tube can be functionally divided into a dorsal domain, with neurons that receive sensory information, and a ventral domain, with motor neurons.
## Clinical findings and epidemiology
Neural tube defects (NTDs) are among the most prevalent of all human congenital anomalies. Fetuses with anencephaly and craniorachischisis typically do not survive to term, although a small proportion of anencephalic infants are viable for a limited time post-parturition. Unlike infants with anterior NTDs, those with meningomyeloceles are viable, yet are likely to suffer from significant disabilities. Worldwide NTDs have a prevalence of approximately 18.6 per 10,000 live births, while the prevalence of NTDs is 6 per 10,000 births in most regions of the United States. There are in excessive of 3,000 NTD-compromised pregnancies annually in the United States, resulting in lifetime medical expenses estimated to exceed $560,000 per infant. Most investigators believe that NTDs have a multifactorial inheritance pattern that involves contributions from environmentaland genetic elements. Data collected over the last 40 years demonstrates that the periconceptional use of folic acid reduces the population burden of NTDs, although the underlying developmental processes that benefit from the folic acid and reduces the NTD risks are not well understood. Clearly, the fortification of food supply did not make the problem of NTDs go away. There remain a large number of NTD cases that are born despite folic acid, with these folic acid-resistant NTDs occurring at an apparent baseline rate of Frontiers in Genetics | www.frontiersin.org 3 May 2021 | Volume 12 | Article 659612 5 per 10,000 live births. Thus, NTDs remain a substantial public health problem even in countries with mandatory folic acid food fortification.
## Nutritional risk factors for ntds
Folate status is clearly established as a modifier of NTD risk, as mothers deficient in water-soluble vitamin B9 are more likely to have infants with NTDs. Folates derived from food are generally in a polyglutamate form, while folic acid is a highly stable synthetic form that is a monoglutamate. The monoglutamate form is initially metabolized in the liver, rather than in the intestines, which is the case for the naturally occurring folates. Since folic acid is physiologically inert in the human body and must be transformed by the enzyme dihydrofolate reductase (DHFR) to bioactive molecules including 5-methyltetrahydrofolate (5MTHF), this process is compromised by the relatively low levels of DHFR in the liver. This results in significant amounts of unmetabolized folic acid entering the systemic circulation. Folates are involved in multiple metabolic functions including transmethylation reactions, the regulation of homocysteine concentrations, and nucleic acid biosynthesis. Several clinically important pharmaceutical compounds with known teratogenic effects, including trimethoprim, Depakote (valproic acid, VPA), and methotrexate, interfere with folate metabolism by inhibiting DHFR, which limits the production of 5MTHF. Folic acid deficiency is not the sole nutrient that, when deficient, has been associated with increased risk for NTDs. Maternal vitamin B12 deficiency is also a known risk factor for NTDs, as vitamin B12 is a co-factor of the enzyme methionine synthase, an important component of one-carbon metabolism (OCM) responsible for converting homocysteine to methionine. In an excellent review by, the results of 24 different studies on the relationship of low maternal serum vitamin B12 levels and risk for NTDs make it clear that low maternal vitamin B12 status increases the NTD risk to the developing embryo. Furthermore, the risk appears to be independent of maternal folate status. While the data is not as robust as it is for folic acid, in the five studies that come from larger cohorts (>80 NTD cases), which are global in nature and involve Canadian, United States, Chinese, and Tunisian populations, low maternal serum vitamin B12 status is linked to increased NTD risk. That said the literature is primarily populated by small studies with limited cohorts of NTD patients. For example,reported on a cohort of 50 Egyptian mothers of infants with a NTD. Maternal serum levels of vitamin B12 were significantly decreased in NTD cases compared to controls, while homocysteine (Hcy) and methylmalonic acid (MMA) concentrations were elevated, demonstrating that low vitamin B12 status is a risk factor for NTDs.reported from the Van province of northern Turkey that 33% of their mothers having infants with NTDs were vitamin B12 deficient. In a study, Fofou-Caillierez et al. (2019) described a cohort with a significant decrease (33.3%) in vitamin B12 concentration along with a 58.6% reduction in SAM among tissues harvested from NTD compared with non-NTD liver tissues. These investigators described a 2.2-fold reduction in vitamin B12 levels in cord blood from NTD fetuses compared to unaffected controls. The decreased vitamin B12 concentration may represent reduced bioavailability in the NTD fetuses or reduced maternal levels. It has also been reported that women consuming diets rich in vitamin B6 and B12, choline, and methionine had lower NTD risks among their progenies. Clearly, a diet rich in one-carbon analytes reduces the risk for birth defects such as NTDs.
## Genetic risk factors for ntds
NTDs are believed to result from multiple factors with both genetic and environmental contributions. Human epidemiological evidence for a genetic component derives from the strong concordance of NTDs between monozygotic twins (7.7%) compared to like sex/dizygotic twins (4.0%;. Furthermore, while neural tube closure defects can be familial after one NTD affected pregnancy, the recurrence risk is 1 in 20, and even after two affected pregnancies, the recurrence risk does not exceed 10%, strongly arguing against a monogenic causation. A number of case-control studies focusing on one, or at most, a few candidate genes have been used to identify alleles suggestive of an association with increased NTD risk. On the strength of folic acid NTD prevention studies, the interrogation of a thermolabile variant (C677T) in the 5-MTHFR gene was proposed to increase the NTD riskin some, but not all, studied cohorts. Candidate genes selected either on the basis of mouse studies or trends in human genome sequences of relatively small case numbers have been replicated in human targeted resequencing studies for a number of genes, including many in the planar cell polarityor WNT signaling pathways.
The well documented over 240 genes, whose mutation cause NTDs in the mouse, support the likelihood that numerous gene defects contribute to NTDs. Mouse genetic studies have also provided the insight that genetic background significantly effects the penetrance of NTDs in individual mice harboring those previously identified mutations and modifier loci that have been mapped in several mutant lines. Most null murine mutants (>90%) present as fetuses with many affected developing organs with high penetrance in homozygotes, while some mutations cause NTDs in digenic, trigenic, and oligogenic combinations, an etiology that is consistent with the genetic causation described in human NTD patients, as human NTDs most often arise through an omnigenic interplay of deleterious genetic variants and environmental factors influencing the function of core pathways such as OCM. What is important to note is that, in Frontiers in Genetics | www.frontiersin.org spite of strong data documenting, the role of genetic factors in the etiology of NTDs, there are no clinically actionable NTD candidate genes known at this time that influence the management of high risk pregnancies. The advent of next generation sequencing (NGS) opens up greater possibilities of dissecting out the genomic architecture underlying NTDs in the coming years.
## Teratogens associated with inducing neural tube defects
## Polycyclic aromatic hydrocarbons
Polycyclic Aromatic Hydrocarbons (PAHs) are commonly found environmental pollutants that are believed to be risk factors for NTDs. PAHs enter the environment following the incomplete burning of biomass and are generally recognized for their grave potential to adversely impact human health. Multiple PAH compounds are considered to be either carcinogenic, mutagenic, and/or teratogenic. Animal experiments have consistently demonstrated that benzo(a)pyrene-7,8-dihydrodiol-9,10epoxide, a i metabolic derivative of benzo(a)pyrene, is capable of producing multiple types of congenital malformations in exposed mouse embryos including: NTDs, gastroschisis, and phocomelia. With respect to human PAH teratogenicity, there have been many epidemiological studies reporting that maternal exposure to PAHs is responsible for an elevated risk of NTDs. In an interesting study conducted in the United States, it was noted that women who are either height and weight proportionate or underweight and are gestationally exposed to PAHs had more NTD affected infants than expected. Another study conducted in Shanxi Province of northern China reported that indoor cooking and heating during the periconceptional period put mothers at an elevated risk for having NTD affected offspring.described a potential association between the concentration of PAHs c in maternal serum and an increased risk for birth defects including NTDs. Their study was based on a case-control design and how the energy usage by households as well as lifestyle parameters impacted PAH exposure. The study involved mothers from Shanxi Province in China who had NTD-complicated pregnancies (n = 117) and 121 control mothers of infants without any malformations. At the time of delivery or pregnancy termination, a blood sample was drawn, and multiple PAHs were analyzed by gas chromatography-mass spectrometry. They determined that the levels of 13 different PAHs differed significantly in the cases than in the controls. A well-defined dose-response relationship was evident between the concentrations of PAHs and the increased risk for an adverse pregnancy outcome such as an NTD. With respect to NTD risk, it was determined that the high-molecular-weight PAHs (H-PAHs) had a greater impact than low-molecular-weight PAHs (L-PAHs). Thus, maternal exposure to PAHs is considered to be a risk factor for NTDs, and that select H-PAHs are associated with a greater NTD risk than are L-PAHs.
A possible association between the aryl hydrocarbon receptor (AHR) and select metabolic enzyme variants as determinants of NTD risk has been under investigation. Cytochrome P450 (CYP) enzymes CYP1A1, CYP1A2, and CYP1B1, which are members of the phase I metabolic enzyme family, are involved in the metabolic activation of PAHs to epoxide intermediates, prior to their conversion into diol-epoxides. There have been a number of single nucleotide polymorphisms (SNPs) in human genes coding for these enzymes that result in significant modifications of their normal enzymatic activities. After collecting blood samples from 534 mothers who conceived newborns or fetuses presenting with NTDs as well as from 534 control mothers who had healthy newborns, they interrogated the samples for 12 polymorphisms in the AHR and cytochrome P450 (CYP) genes. They determined that the CYP1B1 rs2855658 GG variant can modify the effect of indoor air pollution on NTD risk.
The AHR is a transcription factor that is a member of the BHLH superfamily, with a relatively wide and open Ligand Binding Domain (LBD), which can be activated in response to environmental stimuli such as pollutants, xenobiotics, and oxygen levels. Once activated AHR mediates induction of the detoxifying enzymes CYP1A1 and CYP1B1. Intriguingly,established the importance of Pax3 and Pax7, two essential transcription factors required for normal cranial neural crest cell development, on the regulation of the environmental stress response pathway mediated by AHR signaling. Pax 3 variants are well-established risk factors for NTDs. Impacting the expression of critical transcription factors that compromise AHR signaling will no doubt inhibit cellular responses that can compromise normal embryonic development. These results are consistent with the demonstration that aberrant hypermethylation of the Pax3 gene, which leads to its downregulation after PAH exposure, is associated with increased NTD risk in humans.
## Arsenic-induced neural tube defects
Inorganic arsenic (Asi) is a natural environmental contaminant in drinking water, air, and food in the form of arsenate [pentavalent, As (V)] or arsenite [trivalent, As (III)]. Arsenic has many agricultural applications as a pesticide or herbicide, and it is even used therapeutically for the treatment of multiple human diseases. Arsenic is more widely recognized for this toxicity. Asi is believed to be a risk factor for different types of cancer. There is also some evidence that Asi is a human teratogen. Despite the data presented in a number of epidemiological investigations, a universal acceptance of Asi as a source of human congenital malformations is not yet established. As one might imagine, performing such epidemiological studies can be challenging and as a result, the limited number of women with in utero Asi exposure was generally too small to reveal significant associations with specific malformations. Furthermore, most of these human epidemiological investigations required the use of proxy measures of exposure, which could potentially reduce the reliability of the data and lead to subject misclassification.
Acute high dose in utero arsenic exposure is a risk factor for pre-and post-natal mortality. Furthermore, chronic maternal low dose arsenic exposure has been tied to increased pre-and post-natal mortality, as well as low birth weight and developmental disabilities. The small cohort size compromises the ability to statistically demonstrate positive associations with individual birth defects, although multiple investigations revealed an association between maternal arsenic exposure to increased prevalence of congenital malformations in their offspring. A case-control study in Texas focused on maternal heavy metal exposures and birth outcomes revealed trending odds ratios for an increased risk of NTDs specifically with arsenic exposure. Although not statistically significant for reasons previously mentioned, a potential association between maternal arsenic exposure and NTD risk cannot be excluded. There is also indirect evidence that demonstrating As as an NTD risk factor stemming from epidemiological studies of pesticides. Several such studies indicate that NTD risks increase with maternal exposure to pesticides or if pregnant women reside close to agricultural areas. The heightened concern over environmental arsenic as a birth defect risk factor will continue given that arsenic remains to be used both industrially and in agricultural practices.
Much of the ambiguity over human birth defect risks from environmental arsenic exposure has changed over the last two decades. Research conducted globally has provided strong evidence linking maternal arsenic exposure with an increased risk for NTD affected offspring. Studies conducted in Bangladesh have been particularly informative, given that Bangladesh has one of the highest prevalence rates of NTDs in the world, and the large segments of the Bangladeshi population suffers from excessive exposure to arsenic from drinking well water. As arsenic is methylated during its biotransformation, there is a concern that in populations that are highly exposed to arsenic, folate status would be an important confounding variable with respect to NTD prevalence. As the linkage between folic acid and NTDs is well-established, it made sense to look for NTDs in population studies from the same region. They determined that mothers receiving supplemental folic acid significantly lowered the risk for NTDs in the offspring (OR = 0.42, CI 0.18-0.96), thus validating the protective association between folic acid status and NTDs in Bangladeshi infants.
There is a wealth of experimental studies to evaluate the teratogenicity of arsenic in various experimental animal species. These studies utilized multiple arsenic forms, the compounds were administered via differing routes and investigators took advantage of different study designs. What was apparent from these investigations was the very consistency of positive findings of teratogenicity when experimental animals are exposed in utero to arsenic. Most notable among the malformations observed were neural tube and craniofacial defects.reported that oral administration of arsenic to pregnant days significantly increased the rate of exencephaly in mice in the absence of any observed maternal toxicity. There was a distinct dose response for the arsenic induced NTDs, with abnormal fetuses being detected even at the lowest treatment doses of arsenic used in these studies. While these dosages might appear to be extremely high when compared with human environmental exposures such as those in Bangladesh, it is critically important to understand that when conducting laboratory animal developmental toxicity studies, it is prudent to consider working with these higher dosages while being focused on the presence of any maternal toxicity when drawing any meaningful conclusions.
Given the widespread distribution of environmental arsenic, the susceptibility of humans to arsenic toxicity and the fact that orally administered arsenic was teratogenic in the study ofsuggests a potential link between human arsenic exposure and elevated risks for NTDs. This provides sound reason to perform future sophisticated human epidemiological studies to determine if environmental arsenic exposure poses a significant teratogenic threat to exposed human populations as well as genetic interrogations to help to define genetically susceptible population.
## Pesticide-induced neural tube defects
Human exposure to pesticides can occur as a result of either occupational exposure, environmentally, or through food/water consumption of pesticide residues post-application of these compounds. As pesticides are literally designed to be lethal to a range of agricultural pests, they are believed to work through various combinations of toxicological mechanisms, including being endocrine disruptors, immunotoxicants, or neurotoxicants, which tends to target the mammalian nervous system, which is especially sensitive to these agents. Organophosphate pesticides have been shown to be particularly harmful to humans when exposure occurs prenatally or during early childhood. While it is challenging to link NTDs to specific pesticides, many of the organophosphates, chlorpyrifos, and vinclozolin are believed to have such a teratogenic potential. As with other suspected teratogens, the human epidemiological record is seldom consistent or remarkably in depth. Differences in study design and exposure assessment accuracy, which relies on self-reporting, are thought to underlie the different study outcomes, along with limited cohort sizes and complex exposure mixtures. Maternal exposure to endosulfan, DDT, and dichlorodiphenyldichloroethylene (DDE) has been positively associated with an increased risk for NTDs in exposed offspring. Women who have been exposed to these compounds are at an 11-fold increased risk of having an NTD affected pregnancy compared to unexposed control mothers. Clearly, there is much more work to be done with respect to the suspected or confirmed teratogenicity of many agriculturally significant pesticides.
## Maternal hyperthermia-induced neural tube defects
For the past 30 years, there have been several investigations into the role maternal hyperthermia may contribute to the population burden of NTDs. Maternal hyperthermia can be the result of febrile disease, exposure to heat sources occupationally and from the environment, which includes electromagnetic radiation and ultrasound sources. While there is no clear consensus as to the overall impact of maternal hyperthermia on the risk of having an NTD affected pregnancy, the majority of epidemiological studies have reported a positive association. The lack of consistency in the outcome of these studies are likely due to multiple factors, including differing study designs, limited size of the NTD cohorts and heterogeneity of the presenting maternal illness.
It is important to also recognize that in Western Societies, when women have a febrile disease, they are often taking antipyretics and other medications that complicate dissecting out the role of the maternal fever from that of the medications on increasing the risk for NTDs. In one of the larger investigations that relied upon a population-based case control study that included 653 NTDs,determined that elevated risks for NTDs were found when mothers had febrile illnesses during the first trimester. They also reported that when febrile mothers took acetaminophen, the NTD risk was actually lowered . It was also interesting to note that there was a greater risk for anencephaly than for posterior defects.in Beijing using a similar study design as that used in California reported that NTD risks were significantly associated with maternal febrile illness or flu (AOR = 3.93, 95% CI: 2.48-6.23) consistent with the Shaw study. However, these investigators reported that women taking antipyretics for their febrile disease had a higher adjusted odds ratio for anencephaly than for spina bifida. Clearly, maternal fever together with medications used to reduce the fever resulted in an increased risk for NTD affected pregnancies.ascertained a total of 375 NTD cases and 8,247 non-malformed controls for their study cohort. Case mothers were more often overweight or obese to have illnesses during pregnancy that did not induce febrile episodes and to meet the recommended daily folic acid intake when compared to control mothers. Case women who had infants with NTDs reported having febrile disease during pregnancy more commonly (5.1%) than did the mothers of control infants (1.9%;. Among the 2.4-fold increased NTD risk among women suffering from febrile disease while pregnant, it was found that folate replete women had a significantly lower risk for an NTD affected infant than did women with fevers who did not have sufficient folate levels during pregnancy (OR = 3.4; 95% CI: 0.8-4;. Analyzing data obtained from the National Birth Defects Prevention Study, which was multi-year case-control epidemiological survey of congenital malformations in the United States involving telephone interviews of case mothers (n = 17,162) and controls (n = 10,127),determined that there was a significant association with three different NTDs (anencephaly, spina bifida, and encephalocele) and four additional types of birth defects among mothers who reported a fever during early pregnancy. From this study, it was apparent that it was the fever itself, and not the underlying disease, that elevated the risk for NTDs.
The mechanism by which maternal fevers during early gestation disrupt normal NTC has not been adequately resolved to date. There is, however, a significant experimental animal literature related to the impact of maternal hyperthermia on the processes involved in NTC. It is a generally well accepted axiom that maternal core temperature increases above 2°C for long periods of time or increases greater than 4°C for shorter intervals can produce structural malformations in experimental animals. The initial experimental studies were conducted in guinea pigs by an Australian group lead by Prof. Marshall Edwards, who reported miscarriages and newborns with arthrogryposis. The adverse endpoints in experimental model systems were not restricted to arthrogryposis, as hyperthermic exposure can cause the usual spectrum of teratogenic endpoints, including embryolethality, developmental delay/growth retardation, and structural malformations. It has also been observed that maternal hyperthermia exposure is capable of disrupting normal development if the heat exposure is high enough, and it occurs during a sensitive period of development. For example, during early embryogenesis, hyperthermia can alter cellular kinetics and leads to a lack of proliferating cells, delays differentiation of cells, enhances apoptosis, and compromises the developing embryonic vascular system. With respect to NTDs, a brief exposure in mice, rats and guinea pigs beginning prior to and during NTC can produce such malformations. When exposure occurs after the most susceptible period, while it may not induce NTDs, it can still result in adverse pregnancy outcomes. In animal experiments, the congenital defect that is produced depends on the species that is being exposed to heat and precisely when the exposure occurs during morphogenesis. Furthermore, by utilizing inbred mouse strains, it is possible to identify highly susceptible and highly resistant strains, reflective of the genetic background of the given strains. In one notable experimental system, the pregnant dam of different inbred strains was placed in a 50-ml conical tube with holes drilled in the side of the tube to allow water to circulate and then suspended in a 43°C waterbath for 10 min. Depending on the strain/genotype of any given embryo, the response frequency of NTDs would vary widely. For example, the DBA/2J mice were completely resistant to the induction of exencephaly by this heat treatment, while SWV embryos exposed to an identical treatment presented with near 100% penetrant NTDs. By performing linkage studies in an attempt to identify the responsible sensitivity genes, Finnell et al. determined that the sensitivity to hyperthermia induced NTDs was lost in the F1 generation after the first crosses between the resistant C57BL/6 J strain with the SWV strain. Backcrosses to the maternal SWV strain partially restored some of this sensitivity to hyperthermia induced NTDs. Thus, the complex nature of gene-environment interactions requires the development of more sophisticated approaches utilizing the tools of contemporary molecular biology to dissect out significant contributing susceptibility factors to the etiology of environmentally induced NTDs.
# Discussion
## Identification of susceptibility genes associated with anti-seizure medications induced ntds using mouse models
Anti-seizure medications (ASMs) have long been a source of concern when used by pregnant women. The initial association between ASMs and an increased risk for congenital malformations dates back to a short publication in German in 1963 following the recognition of Thalidomide's teratogenic potential. A robust literature that has developed over the last 50 years succinctly documents the apparent teratogenicity of all the frontline ASMs commonly used to manage seizure disorders. These ASMs include phenytointrimethadione, carbamazepine, lamotrigine, levetiracetam, VPA, and topiramate. More alarming is the fact that many of these compounds are now widely prescribed to women of reproductive age for the management of more common afflictions, including neuropathic pain, migraine headaches, mood disorders, obesity, and psychiatric disorders, adding significantly to the number of women of reproductive age who are exposed to these important medications. There are believed to be 1.5 million United States epileptic women of childbearing age responsible for 3-5 infants per 1,000 live born. This is a gross underestimate of the number of women of reproductive age exposed to ASMs annually, given their therapeutic application to treat more prevalent disorders as described above. Recent studies have confirmed that there were in excess of four million prescriptions of anti-seizure medications provided annually to United States women between 15 and 40 years of age. While the prevailing consensus in the literature is that changes in prescribing trends globally has shifted to safer, less teratogenic ASMs, the clinical concern is accordingly lessened. In fact, ASM usage and exposure during pregnancy continues to rise from 15.7 to 21.9 per 1,000 deliveries over a 10-year period ending in 2009. After nearly 60 years of study, there does not appear to be any ASM that is completely free of a teratogenic potential. The literature is replete with studies documenting the teratogenicity of all the commercially available ASMs. Major congenital malformations (MCMs) secondary to in utero ASM exposure is a consistent finding in some but not all exposed infants. The most commonly reported birth defects following maternal use of AEDs include craniofacial, cardiac, skeletal, urologic, and NTDs. A comprehensive review of the teratogenicity of all ASMs is beyond the scope of this review. Attention will be focused on just two compounds-VAP (Depakote) and Topiramate (TPM; Topamax). Depakote is widely utilized worldwide and is associated with the most significant teratogenic threat to developing infants of all the commercially available ASMs. The prospective Neurodevelopmental Effects of Antiepileptic Drugs Study reported that serious adverse pregnancy outcomeseither major structural birth defects or miscarriage occurred in 20% of VPA exposed infants, again validating the consensus that Depakote represents the most teratogenic of the existing frontline ASMs. The International Registry of Antiepileptic Drugs and Pregnancy reported that 18% of the offspring of mothers receiving Depakote in their extensive cohort presented with MCMs in a dose-response manner. It has been estimated that the total costs associated with children exposed in utero to ASMs requiring medical care or educational interventions now exceed $1.8 billion per year. With respect to NTDs and the treatment of women of reproductive age with Depakote, the most important clinical question concerns the benefit of concurrent high does folic acid therapy. To date, with the possible exception of neurocognitive outcomes, there is no evidence demonstrating a protective benefit from high dose folic acid supplementation to reduce the risks of an NTD.
Another clinically important ASM for which there is significant teratogenic concern is Topamax ® [Topiramate; "TPM"; 2,3:4,5-di-O-isopropylidene-(3-D-fructopyranose sulfamate)], a second-generation anti-epileptic medication predominantly not only used in the treatment of seizure disorders but also having therapeutic applications, on and off label, in the treatment of migraine headaches, neurobehavioral disorders, and obesity. Post-marketing surveillance conducted by the pharmaceutical company and advanced information provided by anticonvulsant drug pregnancy registries provided indications that congenital defects, including hypospadias, cleft palate, NTDs, and microcephaly, were being observed in infants exposed in utero to TPM monotherapy. Pregnancy data from the United Kingdom Epilepsy and Pregnancy Registry also revealed infants with cleft lip and palate and hypospadias among 28 informative pregnancies with TPM monotherapy exposure. The major congenital malformation rate consequently was 7.1% and the odds ratio for MCM was 2.75 (95% Cl: 0.62-12.20). TPM's odds ratio for major congenital malformations exceeded the ORs for Tegretol, Lamictal, Dilantin, Neurontin, and Keppra., relying upon pregnancy outcome data from the United Kingdom's Epilepsy and Pregnancy Register, described two cases of oral clefting in a small cohort of infants exposed to TPM monotherapy. The rate was 11-fold greater than that of the general population. There was also a single infant with hypospadias exposed to TPM monotherapy (14-fold increase over the general population), for an overall MCM rate of 4.8%, which is well within the range for most of the frontline anti-seizure medications, as women on ASMs have birth defect rates that exceed those of the general population. Overall, the MCM rate for monotherapy exposure to TPM is similar to firstgeneration ASMs and consistently higher than other secondgeneration ASMs. A final note concerning the teratogenicity of TPM that deserves mention is the recurrence rate of congenital malformations in compromised pregnancies.reported that the recurrence risk of birth defects was 33% for TPM monotherapy and 66% when TPM is used as part of a polytherapy treatment protocol. Although the cohort size of TPM exposed women was small, it is another signal that this medication should be avoided in women considering or who might possibly become pregnant while on the medication. As ASMs are widely prescribed medications, lacking a clear understanding of their teratogenic mechanisms action represents a challenging data gap that limits our ability to safely manage these high-risk pregnancies as well as our ability to design safer and efficacious ASMs. Our laboratory has been actively involved in experiments trying to better understand the geneenvironment interactions that contribute to AED-induced birth defects. The focus of our teratogenic experimentation has been on VPA and its many analogs . In trying to develop appropriate genetic model systems, we have primarily made the use of the inbred mouse strains, SWV/Fnn, which was highly sensitive to VPA-induced NTDs, and C57BL/6J, a resistant strain, where fewer than 10% of the exposed embryos had NTDs. We performed linkage analysis and fine gene mapping experiments in order to identify susceptibility loci responsible for VPA-induced NTDs. Using days from these two highly inbred mouse strains that received sodium valproate (600 mg/kg) via intraperitoneal injection on gestational day E8.5, the embryos failed to close their neural tubes in a strain-dependent manner. Further interrogations of the genomes from 131 backcrossed fetuses with NTDs revealed a major gene mapped to D7Mit285 (p < 2 × 10 −6 ), exceeding the threshold for significant linkage. We determined that recombination events had occurred in the chromosomal region located between D7Mit285 and D7Mit101, which cover a 3.3-Mb region. This helped to establish the presence of a chromosomal region that contained a major gene or genes that regulates susceptibility to VPA-induced NTDs in mice;. More robust genomic tools can now be applied to further refine this region of interest and better define those genetic factors regulating sensitivity to VPA's teratogenicity in mice.
## Understanding the mechanism of action for vpa teratogenicity
The current literature suggests that, while anticonvulsants may share their mechanisms in terms of anti-epileptic and toxic effects, they seem to differ in their mechanisms of teratogenicity, although the latter is largely unknown. Reports in the literature suggest that there are differing mechanisms of action underlying the efficacy for seizure control from that responsible for inducing birth defects. It is likely that the teratogenicity, toxicity, and anticonvulsant effect of VPA are the direct effect of the drug and not its metabolites. Once administered, Depakote is biotransformed into multiple physiologically active compounds; however, given their limited concentration, they do not significantly contribute to the efficacy of the seizure control. What is interesting about the teratogenicity of VPA is the relationship between its potency and the structural requirement that the molecule contain the following: an alpha-hydrogen atom, a carboxyl function and branching on C-2 with two chains containing three carbon atoms each for maximum activity. Several anticonvulsant mechanisms for VPA have been suggested to given its ability to be efficacious for many different epileptic diseases. VPA potentiates GABAergic functions, attenuates amino acidergic neuronal excitation induced by NMDA-type glutamate receptors and alters dopaminergic and serotonergic functions. As far as hepatic metabolism is concerned, VPA inhibits CYP (cytochrome P450) and UDPGT (uridine diphosphate glucuronosyltransferase) enzymes, while the other common anti-epileptic compounds -phenytoin, phenobarbital, primidone, and carbamazepine actually induce the production of these enzymes. Meanwhile, the most commonly used anticonvulsants are eliminated by hepatic metabolism and catalyzed by the enzymes CYP2C9, CYP2C19, CYP3A4, and UDGPT. The teratogenic mechanism for VPA is not well understood. Hypotheses for the teratogenicity of VPA include: interference with folate metabolism, embryonic lipid metabolism, Zn metabolism, neurotransmitter metabolism, altering the methylation of nucleic acids, post-translational methylation, the availability of methyl groups for other important cellular reactions, lowering embryonic pH value, the metabolism of VPA via β-oxidation leading to CoA sequestration, an increase in levels of reactive oxidative stress molecules, and the modulation of chromatin structure secondary to its negative impact on endogenous histone deacetylases. Clearly there have been many possible explanations reported for the teratogenicity of VPA.
VPA exposure is known to increase reactive oxidative species (ROS) production and leads to an increased frequency of homologous recombination. At this time, VPA inhibition of HDACs is believed by many investigators to be the principal way in which the teratogenicity of this anticonvulsant drug is mediated. This inhibition results from the binding to the catalytic center, which restricts substrate access, resulting in and hyper-acetylation of the N-terminal tails of histones H3 and H4 in vitro and in vivo. Inhibition of HDAC results in an overall increase in gene expression. Using Xenopus and zebrafish as model organisms,found that VPA exposure increased neural patterning and cardiac malformations. These defects were observed with transcriptional changes that were closely paralleled by those found in structurally unrelated HDAC inhibitors such as trichostatin A (TSA). VPA and its HDAC inhibiting analogs along with TSA had comparable effects on gene expression across a wide dose range in both model organisms studied, providing strong evidence that VPA exerts its teratogenic effects via HDAC inhibition.
Interaction of VPA with folate metabolism has long been suspected of underlying VPA's teratogenicity and this hypothesis is among the best characterized to date. It has been established that plasma folate and methionine levels are significantly reduced upon VPA treatment, accompanied by an increase in homocysteine and tetrahydrofolate levels. When VPA treatment is accompanied by folate supplementation, the exencephaly rates decreased by 50% in both mice and rats. In humans, as described above, although it is known that folic acid intake can reduce NTDs by 50%, there is no evidence that this is effective in preventing VPA-induced NTDs. There have been several different hypotheses offered with respect to the impact VPA has on folate metabolism. However, one area that has received much less attention is the ability of VPA to directly inhibit the ability of folate receptors to bind and transport folic acid, therefore lowering serum folate concentrations, which may have significant teratogenic consequences.explored the binding affinities of three folate compounds (folic acid, s-folinic acid, and 5-methyltetrahydrofolate) to the folate receptors [folate receptor α (FRα; Folr1), folate receptor β (FRβ; Folr2), and the bovine folate binding protein (bFBP)]. These studies were conducted in both the presence and absence of VPA. The addition of VPA at IC50 concentrations significantly reduces receptor affinity for folates. The non-competitive nature of this interaction with VPA is clear, as increasing the concentration of VPA prevents the receptor from achieving signal saturation. These investigators collected supernatant from HEK293T cells that were previously folate starved and then exposed to either folate or folate and VPA, to see how much of the folates would bind to cell surface folate receptors. As the VPA concentration of VPA was increased, there were significantly less folates bound to the cells.
## Newer methodologies, newer models, and better data
Despite decades of investigation, the etiology of NTDs remains to be clearly elucidated. One of the main reasons for this data gap is the lack of suitable models with which to study early developmental events directly in human embryos. Fortunately, recent technological breakthroughs in the use of neural tube organoids provide a novel three-dimensional (3D) model system that enables us to better understand the development of the human neural tube in an in vitro system. Ideally of course, it is best to test hypotheses in human cells, although this is not always possible. As a result, we have relied upon the use of animal model systems as a reasonable surrogate, although this approach is far from perfect. By using animal models such as the mouse, chick, Xenopus, and zebrafish, investigators have successfully constructed multiple NTD models with which to probe at the cellular level the underlying mechanisms of failed neural tube closure. However, it is important to be cognizant of the fact that the formation of the neural tube includes some differences among different model species, often with respect to the number of initiating closure sites and the timing and sequence of the closure proper. While NTC in mouse embryos closely approximates human NTC, there are still some differences between mouse and human. NTC in human embryos is initiated from only two closure sites, which is equivalent to the closure point 1 and 3 in mice of which there are four closure sites. In chick embryos, there are also two initial closure points, located at the future midbrain and at the hindbrain/cervical boundary. These closure sites undergo a bi-directional closure process. Finally, in Xenopus laevis, NTC occurs almost simultaneously along the entire body axis.
Given these species-specific differences, the use of animal models has their limitations with respect to fully recapitulating the morphogenetic processes involved in human NTC. With the recent emergence of organoid culturing, it is now possible to create in vitro 3D cell models for NTC from human pluripotent stem cells (hPSCs). These neural tube organoids to a great extent simulate the in vivo cell composition of the neural tube and obviate the need to use model organisms for in vivo experimentation. This new approach makes it possible to study human neural tube development utilizing an in vitro culture system. It is also possible to create organoids from genetically modified mouse ESCs, which can serve as important proof of principle studies on specific gene variants. Neural tube organoid culture is based on the self-organizing ability of stem cells grown under extracellular matrix conditions in the presence of essential exogenous signaling factors. This is possible with both hiPSCs/hESCs and murine ESCs. Such cells have been created that maintain not only the proper dorsal-ventral organization, but also rostro-caudal patternsimilar to that of a human neural tube that develops in vivo. Efforts have also been made to use variants of typical cell culturing equipment to essentially create organoids-on-a-chip. These advances offer greater stability of the neural tube phenotype as a result of enhanced precision and control of the in vitro microenvironment. As it is possible to utilize single cells to create neural tube organoids, it opens up a new world of molecular interrogation using single cell RNAseq to better understand the underlying changes at the genomic level, leading to the failure of neural tube closure. This will be an essential tool to developing intervention strategies to not only prevent preventable birth defects, but also to create essential therapeutic approaches to minimizing the morbidities associated with spina bifida. Finally, the importance of environmental influences on the 3D organization of DNA cannot be overlooked. Topologically associating domains (TADs) are genomic regions that represent potentially compromised structures that can be altered by a range of teratogens and are amenable to future investigations that may change the way we have been thinking about environmentally-induced birth defects.
## Concluding remarks
NTDs represent one of many excellent examples of complex congenital malformations that result for as yet undefined interactions between genes and environmental teratogens. Efforts to define such interactions and clearly identify the sensitivity or susceptibility genes have been slow to evolve. However, recent development of advanced DNA sequencing tools at reasonable experimental costs have created new possibilities that this vexing data gap might soon be resolved. Like many investigators, we look forward to the continued evolution of scientific technologies and approaches that help us to understand the genetic architecture of NTDs and biomechanical events responsible for these devastating birth defects. We look forward to the day when preventable birth defects can indeed be prevented.
# Author contributions
RF and CC primarily written the manuscript. RF, BW, XC, YL, JS, S-EK, GT, YL, and RC participated in editing and revising the draft manuscript. RF, BW, RC, XC, and YL generated the data for the manuscript. RC, XC, S-EK, and GT generated the figures. All authors contributed to the article and approved the submitted version.
# Funding
This work was supported by the NIH grants HD081216, HD083809, HD067244, HD093758, HD095520, HD100535, and T32 ES027801. Dr. Steele is supported by a fellowship from the T32 training grant. |
Global variations in mortality in adults after initiating antiretroviral treatment: an updated analysis of the International epidemiology Databases to Evaluate AIDS cohort collaboration
for the International epidemiology Databases to Evaluate AIDS (IeDEA) Collaboration Background: UNAIDS models use data from the International epidemiology Databases to Evaluate AIDS (IeDEA) collaboration in setting assumptions about mortality rates after antiretroviral treatment (ART) initiation. This study aims to update these assumptions with new data, to quantify the extent of regional variation in ART mortality and to assess trends in ART mortality.Methods: Adult ART patients from Africa, Asia and the Americas were included if they had a known date of ART initiation during 2001-2017 and a baseline CD4 þ cell count. In cohorts that relied only on passive follow-up (no patient tracing or linkage to vital registration systems), mortality outcomes were imputed in patients lost to follow-up based on a meta-analysis of tracing study data. Poisson regression models were fitted to the mortality data.Results: 464 048 ART patients were included. In multivariable analysis, mortality rates were lowest in Asia and highest in Africa, with no significant differences between African regions. Adjusted mortality rates varied significantly between programmes within regions. Mortality rates in the first 12 months after ART initiation were significantly higher during 2001-2006 than during 2010-2014, although the difference was more substantial in Asia and the Americas [adjusted incidence rate ratio (aIRR) 1.43, 95% CI: 1.22-1.66] than in Africa (aIRR 1.07, 95%
# Introduction
Antiretroviral treatment (ART) is estimated to have reduced the number of AIDS deaths globally by 6.6 million in the period up to 2013, and in many of the countries most severely affected by the HIV epidemic, ART has had a profound demographic impact [bib_ref] Mortality trends in the era of antiretroviral therapy: evidence from the Network..., Reniers [/bib_ref] [bib_ref] Estimating the impact of antiretroviral treatment on adult mortality trends in South..., Johnson [/bib_ref]. However, HIV case surveillance and vital registration systems are often weak or absent in the most severely affected countries, necessitating mathematical models to estimate AIDS mortality and the impact of ART. The Joint United Nations Programme on HIV/AIDS (UNAIDS) supports the development and application of the Spectrum tool for this purpose [bib_ref] Updates to the Spectrum/Estimations and Projections Package model for estimating trends and..., Stover [/bib_ref] ; this tool is also widely used to estimate other HIV indicators and to evaluate the impact and cost-effectiveness of different interventions [bib_ref] What is required to end the AIDS epidemic as a public health..., Stover [/bib_ref]. AIDS mortality estimates produced by Spectrum and other mathematical models depend on assumptions about mortality rates after ART initiation. UNAIDS has previously relied on the International epidemiology Databases to Evaluate AIDS (IeDEA) Collaboration to provide estimates of mortality of patients receiving ART in different regions to parameterize the Spectrum model [bib_ref] All-cause mortality in HIV-positive adults starting combination antiretroviral therapy: correcting for loss..., Anderegg [/bib_ref] [bib_ref] Estimated mortality of adult HIV-infected patients starting treatment with combination antiretroviral therapy, Yiannoutsos [/bib_ref]. Previous IeDEA analyses to estimate Spectrum parameters incorporated mortality data from patients starting ART in the pre-2011 [bib_ref] Estimated mortality of adult HIV-infected patients starting treatment with combination antiretroviral therapy, Yiannoutsos [/bib_ref] and 2009-2014 [bib_ref] All-cause mortality in HIV-positive adults starting combination antiretroviral therapy: correcting for loss..., Anderegg [/bib_ref] periods. These analyses were limited in their ability to estimate mortality of patients starting ART at higher CD4 cell counts, as such patients only became eligible for ART in low-income and middle-income countries after the WHO recommended universal ART eligibility in 2015. The analyses conducted for UNAIDS have also not assessed whether there have been temporal changes in ART mortality (after controlling for differences in baseline CD4 cell count and ART duration), although some studies suggest that ART mortality rates have declined significantly [bib_ref] Mortality and loss to follow-up among HIVinfected persons on long-term antiretroviral therapy..., Carriquiry [/bib_ref] [bib_ref] Improved survival in HIV treatment programmes in Asia, De La Mata [/bib_ref]. A further concern is that previous analyses applied multiplicative 'correction factors' to observed mortality rates to correct for under-ascertainment of mortality in patients lost to follow-up (LTFU), but these correction factors might not have adequately reflected heterogeneity in ascertainment of mortality across settings, as they were based only on data from South Africa and Kenya.
This study aims to update adult ART mortality estimates for the Spectrum model, incorporating more recent IeDEA data (up to 2017) and advancing the mortality estimation methods. This study also aims to assess trends in adult ART mortality, to quantify mortality levels since introduction of universal ART eligibility, to evaluate the extent of inter-regional variation in mortality, and to assess whether inter-regional differences are explained by known predictors of mortality.
# Methods
IeDEA is an international collaboration of ART programmes, divided into seven regions: Asia-Pacific; Central America, South America and the Caribbean (hereafter 'Latin America'); North America; Central Africa; East Africa; Southern Africa and West Africa [bib_ref] Cohort Profile: the international epidemiological databases to evaluate AIDS (IeDEA) in sub-Saharan..., Egger [/bib_ref] [bib_ref] IeDEA-WHO Research-Policy Collaboration: contributing real-world evidence to HIV progress reporting and guideline..., Zaniewski [/bib_ref]. Participating programmes are embedded in routine care services, with frequencies of scheduled clinic visits varying across programmes, in line with local guidelines. Each region has an independent data centre that pools, deidentifies and analyses the data. Each programme participating in this study obtained ethical approval from relevant local institutions to collect and share patient data; in addition, each regional data centre obtained ethical approval to collate and analyse the de-identified data.
The analysis included adults (aged 15 years and older) who started ART between 2001 and 2017 and were ART-naïve at enrolment (or who had a date of first ART initiation if not ART-naïve), and who had a recorded CD4 cell count in the six months prior to starting ARTor two weeks after starting ART. ART was defined as a combination of at least three antiretroviral drugs. Consistent with previous UNAIDS analyses [bib_ref] All-cause mortality in HIV-positive adults starting combination antiretroviral therapy: correcting for loss..., Anderegg [/bib_ref] [bib_ref] Estimated mortality of adult HIV-infected patients starting treatment with combination antiretroviral therapy, Yiannoutsos [/bib_ref] , we defined baseline CD4 cell count as the CD4 cell count closest to the time of ART initiation and we defined patients as LTFU if their last visit date was more than six months prior to the database closure without any record of other outcomes (death or transfer). To estimate parameters in the Spectrum model, we used the same covariate categories as the Spectrum model: baseline CD4 cell count was categorized as 0-49, or !500 cells/ml; time since ART initiation ('duration') was categorized as 0-5 months, 6-11 months or !12 months; and age was categorized as or !45 years. We conducted sensitivity analyses to assess the effect of including patients with missing baseline CD4 values, using multiple imputation to assign baseline CD4 cell counts, and to assess the effect of modelling age effects using restricted cubic splines rather than a categorical approach [bib_ref] Assessing and interpreting the association between continuous covariates and outcomes in observational..., Shepherd [/bib_ref]. Except in the case of sub-Saharan Africa (discussed below), follow-up time was defined from the date of ART start (or date of enrolment if later) to the date of death or the last date that the patient was known to be alive. The resulting mortality estimates are referred to as 'ART mortality rates' although some patients might not have been on ART at the time of death.
Because most patients starting ART at higher CD4 cell counts prior to 2015 were doing so because they met clinical rather than immunological ART eligibility criteria, and because such patients are not representative of asymptomatic individuals with higher CD4 cell counts, we excluded individuals who qualified for ART based solely on clinical criteria. In the Latin American and North American programmes, as well as in some Asian programmes, this meant excluding patients who had a clinical diagnosis of AIDS prior to starting ART, if they started ART with a CD4 cell count above the CD4 eligibility threshold that applied at the time. In the sub-Saharan African programmes and some Asian programmes, in which the recording of baseline clinical stage was incomplete, patients were excluded if their baseline CD4 cell count was above the CD4 eligibility threshold at the time of starting ART (on the assumption that they qualified for ART based only on clinical eligibility criteria). Details of the eligibility criteria that applied in each country in each year were obtained from previous reviews of ART eligibility criteria [bib_ref] When will sub-Saharan Africa adopt HIV treatment for all?, Gupta [/bib_ref] [bib_ref] Realizing the potential of treatment as prevention: global ART policy and treatment..., Gupta [/bib_ref] [bib_ref] HIV treatment eligibility expansion and timely antiretroviral treatment initiation following enrollment in..., Tymejczyk [/bib_ref] and from consultation with local experts; full details are provided in Supplementary Table S1, http://links.lww.com/QAD/B527. A sensitivity analysis was conducted to assess the effect of not applying these exclusion criteria.
Programmes differed in their approach to ascertaining mortality. In the South African programmes and most North and Latin American programmes, patient records were regularly linked to vital registration systems, which were judged to have high completeness [bib_ref] A comparison of death recording by health centres and civil registration in..., Johnson [/bib_ref] [bib_ref] Estimating the completeness of death registration: an empirical method, Adair [/bib_ref] [bib_ref] What can we conclude from death registration? Improved methods for evaluating completeness, Murray [/bib_ref]. In some North and Latin American programmes, and most Asian programmes, attempts were made to contact patients who missed visits, either by telephone or by home visits ('active follow-up') [bib_ref] Mortality and loss to follow-up among HIVinfected persons on long-term antiretroviral therapy..., Carriquiry [/bib_ref] [bib_ref] Loss to follow-up trends in HIV-positive patients receiving antiretroviral treatment in Asia..., De La Mata [/bib_ref] [bib_ref] Closing the gap: increases in life expectancy among treated HIV-positive individuals in..., Samji [/bib_ref] , and this was also judged to yield reasonably accurate estimates of mortality. However, in most sub-Saharan African programmes, mortality was ascertained passively (no active follow-up or linkage to vital registration systems), and the high proportion of patients LTFU suggested substantial unrecorded mortality [bib_ref] Retention and mortality on antiretroviral therapy in sub-Saharan Africa: collaborative analyses of..., Haas [/bib_ref]. To address this problem, we used an imputation model to simulate mortality outcomes in the six months after LTFU in sub-Saharan African programmes (excluding South Africa) [bib_ref] Adjusting mortality for loss to follow-up: analysis of five ART programmes in..., Brinkhof [/bib_ref]. Suppose T i is the time to death (in days after the last visit) for patient i who is classified LTFU. We assume T i is Weibull-distributed with parameters l i and f, where l i ¼ x i b (x i representing the covariate information for individual i and b representing the covariate effects on the mortality rate). We chose the Weibull distribution over the more flexible generalized gamma distribution as the Weibull distribution is easier to invert, which is important for simulation purposes. We estimated the f and b coefficients by fitting the Weibull model to mortality data from an individual patient data meta-analysis of studies that traced LTFU patients in African ART programmes [bib_ref] Outcomes of patients lost to follow-up in African antiretroviral therapy programs: individual..., Chammartin [/bib_ref] , augmented by more recent data from a large Zambian tracing study [bib_ref] Estimated mortality on HIV treatment among active patients and patients lost to..., Holmes [/bib_ref]. For each individual who was LTFU, we sampled a value t i from the Weibull distribution for T i . If t i was 180 days or more, follow-up was censored at the last visit date þ 180 days; otherwise the date of death was set to the last visit date þ t i . For each LTFU patient, we simulated five outcomes, thus generating five datasets for the sub-Saharan African cohorts that we combined using Rubin's rules. We limited the simulation of mortality outcomes to the first six months after last contact due to the limited numbers of deaths at longer tracing durations and the resulting uncertainty in the extrapolation of the Weibull hazard to longer durations.
Consistent with previous UNAIDS analyses [bib_ref] All-cause mortality in HIV-positive adults starting combination antiretroviral therapy: correcting for loss..., Anderegg [/bib_ref] [bib_ref] Estimated mortality of adult HIV-infected patients starting treatment with combination antiretroviral therapy, Yiannoutsos [/bib_ref] , we used Poisson regression models to estimate average mortality rates for each covariate category in Spectrum. We fitted separate regression models for the first 12 months after ART initiation and for durations more than 12 months after ART initiation, to account for nonproportional hazards. We also ran separate regression models for the African and non-African regions, due to differences between generalized and concentrated HIV epidemic settings in both overall levels of mortality as well as sociodemographic determinants of mortality. We included region-specific effects in each model, and used random effect models (with random intercepts) to account for variation across programmes within each region. The ratio of mortality at the 90th percentile of random effects to that at the 10th percentile of random effects was calculated to demonstrate the extent of interprogramme variation in mortality within regions, assuming that random effects were normally distributed [bib_ref] Heterogeneity in outcomes of treated HIVpositive patients in Europe and North America:..., May [/bib_ref]. We treated South Africa as a separate 'region' because of the difference in mortality ascertainment in this country (relative to the rest of sub-Saharan Africa) and because of the large number of ART patients in this country. The West and Central African regions were combined due to the small numbers of patients in Central Africa, but in a sensitivity analysis we included separate effects for Central Africa. All statistical analyses were performed using STATA 15.1 (StataCorp, College Station, TX, USA). In the Spectrum model, IeDEA estimates of all-cause mortality in ART patients were converted into estimates of HIV-related mortality by subtracting United Nations Population Division estimates of non-HIV mortality in each region(more detail is provided in Supplementary material Section 2, http://links.lww.com/QAD/ B527). In the results that follow, we present only the IeDEA estimates of all-cause mortality.
# Results
846 418 adults started ART in 2001-2017, of whom we excluded 271 959 (32%) due to missing baseline CD4 values, 12 541 (1%) due to missing or invalid outcome dates, and 97 870 (12%) who qualified for ART solely on clinical criteria. Our main analysis therefore included 464 048 ART patients, from 72 different IeDEA programmes, the majority from sub-Saharan Africa (408 131, 88%). Demographic characteristics differed substantially across regions: the proportion of patients who were female ranged from 16% in North America to 65% in West and Central Africa, while the proportion of patients aged 45 years or older at the time of ART initiation ranged between 17% in Southern Africa and 41% in North America [fig_ref] Table 1: Patient characteristics at antiretroviral treatment initiation, 2001-2017 [/fig_ref]. Baseline immunological status also differed substantially across regions, with the percentage starting ART at a CD4 cell count <200 cells/ ml ranging between 33% in North America and 78% in South Africa. In the North American cohort a substantial fraction of patients (12%) acquired HIV following needle sharing.
The imputation model of mortality after LTFU in African tracing studies was based on 4751 adult patients for whom a vital status could be established (characteristics of the tracing studies and traced patients are included in Supplementary Tables S10 and S11, http://links.lww. com/QAD/B527, respectively). Of these, 77% were from Southern Africa, 18% were from East Africa and 5% were from Central Africa. The results of the Weibull regression model are shown in [fig_ref] Table 2: The model was fitted to data from 4751 adult patients traced after... [/fig_ref]. Mortality risk among patients LTFU was significantly associated with older age, lower baseline CD4 cell count and shorter ART duration, but the effects of year and sex were not statistically significant. The Weibull shape parameter was 0.60 (95% CI: 0.57-0.63), indicating a strong negative relationship between time since LTFU and the mortality rate. The imputation model estimated that 57% of all deaths on ART in sub-Saharan Africa [fig_ref] Figure 1: Cumulative mortality hazard, by region [/fig_ref] shows the cumulative all-cause mortality estimates, after imputing mortality outcomes for the African cohorts in which there was no active follow-up or vital registration linkage. In the non-African cohorts, AIDS 2019, Vol 33 (Suppl 3) mortality rates appeared relatively low in North America at shorter ART durations, but at longer durations cumulative mortality rates were higher than those in Latin America and Asia-Pacific. Cumulative mortality rates were consistently higher in the sub-Saharan African cohorts, with mortality rates in the African region being lowest in South Africa and highest in West and Central Africa.
In the multivariable analysis, mortality differences between regions remained significant [fig_ref] Table 3: Multivariable analysis of mortality after antiretroviral treatment initiation [/fig_ref]. Mortality in Latin America was roughly double that in Asia-Pacific, and although mortality in North America was not significantly different from that in Asia-Pacific during the first 12 months of ART, it was more consistent with that observed in Latin America at longer ART durations. Mortality rates did not differ significantly between African regions in the multivariable analysis, although in a sensitivity analysis in which Central and West Africa were considered separately, mortality appeared significantly lower in Central Africa than in the other African regions, during the first 12 months of ART [fig_ref] Table 1: Patient characteristics at antiretroviral treatment initiation, 2001-2017 [/fig_ref] , http://links.lww.com/QAD/B527). There was substantial variation in mortality between programmes within each region. In the non-African regions, after controlling for region effects and other predictors, the mortality rate at the 90th percentile of random effects was 5.1 times that at the 10th percentile (exp(0.64 Â 2 Â 1.28), where 1.28 is the 90th percentile of the standard normal distribution). Heterogeneity was lower in the African cohorts, with the 90-10th percentile ratio being 2.9 in the first 12 months of ART and 3.2 at longer ART durations.
Mortality rates in the first 12 months after ART initiation appeared to decline over time in the non-African cohorts, but changed relatively little in the African cohorts [fig_ref] Table 3: Multivariable analysis of mortality after antiretroviral treatment initiation [/fig_ref] [fig_ref] Table 1: Patient characteristics at antiretroviral treatment initiation, 2001-2017 [/fig_ref] , http:// links.lww.com/QAD/B527).
The effect of baseline CD4 cell count on mortality was more significant during the first 12 months after ART initiation than at longer ART durations, and mortality risks decreased as baseline CD4 cell counts increased [fig_ref] Table 3: Multivariable analysis of mortality after antiretroviral treatment initiation [/fig_ref]. However, in the sensitivity analysis in which patients were not excluded if their CD4 cell count was above the ART eligibility threshold at the time of ART initiation, the incidence rate ratios were in most cases closer to 1, suggesting a weaker negative relationship between baseline CD4 cell count and mortality at higher CD4 cell counts [fig_ref] Table 4: Effects of baseline CD4 R cell count [/fig_ref]. Results did not change substantially when CD4 cells counts were imputed for those individuals with missing baseline CD4 values, although in African cohorts the imputed CD4 cell counts appeared less strongly predictive of mortality during the first 12 months after ART initiation (Tables S15 and S16, http://links.lww.com/QAD/B527). Results also remained largely unchanged when alternative models of the age effect were considered (Tables S17 and S18, http://links.lww.com/QAD/B527), and when ART
## S288
AIDS 2019, Vol 33 (Suppl 3) eligibility criteria were controlled for (Tables S19 and S20, http://links.lww.com/QAD/B527).
# Discussion
The current study shows evidence of substantial variation in mortality in treated HIV-positive adults, both across global regions and within regions. Consistent with previous studies of intercohort variation in mortality [bib_ref] Heterogeneity in outcomes of treated HIVpositive patients in Europe and North America:..., May [/bib_ref] [bib_ref] Mortality in patients with HIV-1 infection starting antiretroviral therapy in South Africa,..., Boulle [/bib_ref] , we show that this variation cannot be explained only in terms of factors such as differences in baseline CD4 cell count, injecting drug use or completeness of mortality ascertainment, although differences observed between sub-Saharan African regions [fig_ref] Figure 1: Cumulative mortality hazard, by region [/fig_ref] ceased to be significant in the multivariable analysis. Possible explanations for residual variation in mortality include differences across programmes in the socioeconomic profiles of their patient populations, differences in facility type (e.g. in some settings tertiary centres may tend to treat sicker patients [bib_ref] Better antiretroviral therapy outcomes at primary healthcare facilities: an evaluation of three..., Fatti [/bib_ref] , differences across regions in ART monitoring strategies (with virological monitoring generally yielding better mortality outcomes than clinical or immunological monitoring alone [bib_ref] Outcomes of antiretroviral treatment in programmes with and without routine viral load..., Keiser [/bib_ref] [bib_ref] Monitoring of antiretroviral therapy and mortality in HIV programmes in Malawi, South..., Estill [/bib_ref] , differences in the effectiveness and tolerability of the locally Global mortality variations in antiretroviral treatment patients Johnson et al. S289 . Effect of calendar period on mortality rates more than 12 months after antiretroviral treatment initiation, before and after controlling for differences in mortality by duration. [fig_ref] Table 3: Multivariable analysis of mortality after antiretroviral treatment initiation [/fig_ref].
recommended drug regimens, differences in levels of antiretroviral drug resistance [bib_ref] Global trends in antiretroviral resistance in treatmentnaive individuals with HIV after rollout..., Gupta [/bib_ref] [bib_ref] Global epidemiology of drug resistance after failure of WHO recommended first-line regimens..., Tenores Study Group [/bib_ref] and differences in resources for patient tracking and the diagnosis and management of comorbidities.
The unusually high estimates of mortality in sub-Saharan Africa [fig_ref] Figure 1: Cumulative mortality hazard, by region [/fig_ref] and [fig_ref] Table 3: Multivariable analysis of mortality after antiretroviral treatment initiation [/fig_ref] could be attributed to a number of factors. Tuberculosis accounts for almost half of all deaths in HIV-positive individuals in Africa [bib_ref] Prevalence of tuberculosis in post-mortem studies of HIV-infected adults and children in..., Gupta [/bib_ref] , but is markedly less prevalent in high-income settings. Poor access to cotrimoxazole and isoniazid prophylaxis in resource-limited settings could also contribute to excess mortality associated with tuberculosis and other HIVrelated diseases [bib_ref] Implementation of co-trimoxazole prophylaxis and isoniazid preventive therapy for people living with..., Date [/bib_ref]. Non-HIV mortality, which accounts for much mortality in ART patients, is also substantially higher in the sub-Saharan African region than in other regions.
The unusual mortality pattern in North America (lower than in other concentrated epidemic settings at early ART durations, but higher at later durations) is consistent with previous inter-regional comparisons [bib_ref] Mortality in patients with HIV-1 infection starting antiretroviral therapy in South Africa,..., Boulle [/bib_ref] [bib_ref] Mortality differences after ART initiation in HIV-positive women from Europe, the Americas..., Jarrin I ; Iedea [/bib_ref] , and could be partly because patients are only included in the North American database if they have at least two visits in the year of enrolment [bib_ref] Laboratory measures as proxies for primary care encounters: implications for quantifying clinical..., Rebeiro [/bib_ref] , implying a selection bias due to the exclusion of individuals who die or transfer care before their second visit. The higher rates of mortality in North American patients compared with Asian patients, at longer ART durations, is consistent with a recent analysis that found significantly lower viral suppression in North American patients compared with Asian patients [bib_ref] HIV viral load suppression in adults and children receiving antiretroviral therapy -results..., Jiamsakul [/bib_ref]. This may be due to the nature of the American healthcare system, with patients frequently transferring between public and private care, and the resulting instability in care contributing to higher long-term mortality [bib_ref] Sex, race, and HIV risk disparities in discontinuity of HIV care after..., Rebeiro [/bib_ref]. The North American patients are also older on average than those in other regions, although controlling for age in different ways does not appear to change the relative difference between North America and other regions (Tables S17 and S18, http:// links.lww.com/QAD/B527).
Even within regions, substantial variation in mortality is observed, with the ratio of the adjusted mortality at the 90th percentile of random effects to that at the 10th percentile being roughly 5 in concentrated epidemic settings and roughly 3 in sub-Saharan African settings. The slightly lower heterogeneity in the sub-Saharan African regions is probably due to the same model being used to impute mortality outcomes across all cohorts, which may lead to the true extent of the heterogeneity being understated. It is important that mathematical models of the impact of ART reflect the uncertainty regarding the ART mortality rates that apply locally. Ideally models should be calibrated to local mortality data to reduce this uncertainty [bib_ref] Estimating the impact of antiretroviral treatment on adult mortality trends in South..., Johnson [/bib_ref] [bib_ref] Improvements in Spectrum's fit to program data tool, Mahiane [/bib_ref] , but when such data are not available and these IeDEA estimates are used, confidence intervals around model outputs should reflect the standard deviations of the random effects terms estimated here. Future analyses should aim to reduce the variation of these random effects, for example by regressing on country GDP per capita and on rates of viral suppression, factors that are likely to account for much of the variation in mortality within regions.
This study suggests that mortality rates in ART patients have declined over time in Asia and the Americas, even after controlling for improvements in baseline characteristics and changes in treatment duration. This is consistent with declines previously observed in concentrated epidemic settings [bib_ref] Mortality and loss to follow-up among HIVinfected persons on long-term antiretroviral therapy..., Carriquiry [/bib_ref] [bib_ref] Improved survival in HIV treatment programmes in Asia, De La Mata [/bib_ref]. However, in sub-Saharan African cohorts the reductions in mortality over time are only noticeable when considering follow-up more than 12 months after ART initiation, and when considering a more detailed model of duration effects, even these declines appear questionable after 2006 . South African data, which are based on more robust mortality ascertainment, also suggest little or no decline in treated mortality over time [bib_ref] Twelve-year mortality in adults initiating antiretroviral therapy in South Africa, Cornell [/bib_ref] [bib_ref] Life expectancy trends in adults on antiretroviral treatment in South Africa, Johnson [/bib_ref]. Declines in mortality might be expected, given innovations in service delivery (such as adherence clubs [bib_ref] Effectiveness of patient adherence groups as a model of care for stable..., Luque-Fernandez [/bib_ref] , the introduction of newer drugs that are better tolerated and more effective in suppressing HIV [bib_ref] Comparative efficacy and safety of first-line antiretroviral therapy for the treatment of..., Kanters [/bib_ref] , and the increasing use of fixeddose combination ART [bib_ref] Systematic review and meta-analysis: Patient and programme impact of fixed-dose combination antiretroviral..., Ramjan [/bib_ref]. Declines may also be due to improvements in baseline clinical stage, which we did not control for in this analysis because the information was missing in many cohorts, and because the Spectrum model (in common with most other mathematical models [bib_ref] HIV treatment as prevention: systematic comparison of mathematical models of the potential..., Eaton [/bib_ref] does not include a separate stratification by clinical stage. The absence of a strong decline in mortality in African cohorts may be related to declining visit frequencies [bib_ref] Extending visit intervals for clinically stable patients on antiretroviral therapy: multicohort analysis..., Haas [/bib_ref] , or increasing rates of treatment interruption [bib_ref] Treatment interruption in a primary care antiretroviral therapy program in South Africa:..., Kranzer [/bib_ref] , which may have offset the expected gains from therapeutic improvements. It is also possible that the relatively rapid growth in drug resistance in East and Southern Africa [bib_ref] Global trends in antiretroviral resistance in treatmentnaive individuals with HIV after rollout..., Gupta [/bib_ref] and the relatively high prevalence of tenofovir drug resistance in sub-Saharan Africa [bib_ref] Global epidemiology of drug resistance after failure of WHO recommended first-line regimens..., Tenores Study Group [/bib_ref] may be part of the reason why mortality in sub-Saharan Africa has not declined to the same extent as in other regions. Alternatively, the slow mortality decline may be due to the parameterization of the imputation model, which suggested only a modest decline over time in mortality in LTFU patients [fig_ref] Table 2: The model was fitted to data from 4751 adult patients traced after... [/fig_ref] , in contrast to the significant reduction in the original meta-analysis on which the imputation model was based [bib_ref] Outcomes of patients lost to follow-up in African antiretroviral therapy programs: individual..., Chammartin [/bib_ref]. Further research is required to determine the reasons for the relatively modest declines in treated mortality rates in the sub-Saharan African region.
This analysis advances previous parameterizations of the Spectrum model in a number of ways. In addition to incorporating more recent data and assessing time trends in ART mortality, this analysis adopts an innovative approach to correcting for unascertained mortality after LTFU, recognizing that the extent of the underascertainment of mortality is likely to differ substantially across settings. The previous analysis applied the same correction factors in all regions, leading to assumed mortality rates being roughly double those observed in IeDEA cohorts [bib_ref] All-cause mortality in HIV-positive adults starting combination antiretroviral therapy: correcting for loss..., Anderegg [/bib_ref]. In the present analysis, no correction factors are applied in the North American, Latin American and Asia-Pacific datasets, with the result that mortality rates are substantially lower than estimated previously. Although the use of an imputation model (in place of correction factors) has not substantially changed the overall levels of mortality in the African regions, the major advantage of the imputation model is that adjustments are applied at an individual level rather than at an aggregated or cohort level, which makes it possible to estimate the effects of covariates on mortality with greater accuracy.
A further methodological innovation is that we have excluded patients who started ART if they qualified for ART based only on clinical criteria. Although this does not materially change estimates of mortality in patients with low baseline CD4 þ cell counts (as such patients qualify for ART regardless of their clinical stage), it does lead to somewhat lower mortality rates in patients starting ART at higher CD4 þ cell counts [fig_ref] Table 4: Effects of baseline CD4 R cell count [/fig_ref] and thus more substantial modelled benefits from early ART initiation. This is consistent with evidence of the clinical benefits of early ART in randomized controlled trials [bib_ref] Initiation of antiretroviral therapy in early asymptomatic HIV infection, Insight Start Study [/bib_ref]. The exclusion of individuals who qualified for ART based only on clinical criteria is important as these individuals are over-represented in high baseline CD4 þ cell count categories, particularly in countries that have only recently moved to universal ART eligibility. These individuals also have relatively high mortality rates, making them unrepresentative of the general population of patients starting ARTat higher CD4 þ cell counts in the era of universal ART eligibility.
This analysis has some limitations. Although most of the programmes in the Asia-Pacific region relied on active follow-up to ascertain outcomes, it is unclear how consistently it was applied, with the result that there may be some underestimation of mortality rates. However, data from Thailand and Indonesia suggest that deaths after LTFU account for a relatively small fraction of total deaths in these settings [bib_ref] Life expectancy after initiation of combination antiretroviral therapy in Thailand, Teeraananchai [/bib_ref] [bib_ref] Predictors of mortality among patients lost to follow up antiretroviral therapy, Dewi [/bib_ref] , and other studies from North America and Latin America have found only modest differences in mortality between programmes that employ active follow-up and those that rely on linkage to vital registration systems [bib_ref] Mortality and loss to follow-up among HIVinfected persons on long-term antiretroviral therapy..., Carriquiry [/bib_ref] [bib_ref] Closing the gap: increases in life expectancy among treated HIV-positive individuals in..., Samji [/bib_ref]. Another limitation is that we have pooled data across IeDEA regions rather than fitting separate regression models for each region, which may have led to some inter-regional differences in covariate effects being obscured. Although initial attempts were made to fit the regression models separately for each region, this led in some cases to implausible point estimates with extremely wide CIs, which were judged to not be appropriate as inputs for Spectrum. A further limitation is that we lack information on causes of death in many IeDEA cohorts, and thus cannot assess the extent to which inter-regional differences in all-cause mortality might be driven by specific diseases such as tuberculosis, or non-HIV-related causes. The Poisson regression approach imposes an assumption of piecewise-constant mortality rates, which may be appropriate for the purpose of estimating parameters in the Spectrum model, but which may lead to biased estimates of the effects of covariates on mortality (as demonstrated in , which compares the effects of using shorter duration intervals, and Tables S21 and S22, which compare the effects of fitting more flexible Cox proportional hazards models). Changes in mortality over the first 6 months of ART are particularly dramatic [bib_ref] Modeling AIDS survival after initiation of antiretroviral treatment by Weibull models with..., Yiannoutsos [/bib_ref] , but the Spectrum model requires a single parameter to represent average mortality over this treatment duration. Similarly, our analysis finds evidence that ART mortality continues to decline for durations greater than 24 months on ART [fig_ref] Table 1: Patient characteristics at antiretroviral treatment initiation, 2001-2017 [/fig_ref] , http://links.lww.com/QAD/B527), but in the primary analysis we estimated a single mortality rate for more than 12 months after ART initiation, for consistency with the current Spectrum model structure.
Further limitations relate to the large fraction of deaths among persons LTFU that must be imputed in African settings, and the limited and imperfect data to inform the imputation model. The imputation model relied only on data from traced patients whose vital status could be established, but because a substantial proportion of LTFU patients could not be traced [bib_ref] Outcomes of patients lost to follow-up in African antiretroviral therapy programs: individual..., Chammartin [/bib_ref] , and because the sampling of LTFU patients to be traced is not always random, mortality estimates may be biased. Due to the limited amount of tracing study data from outside Southern Africa, it was not feasible to include region in the imputation model, although this covariate was included in the main analysis. This might have led to some bias in Rubin's variance estimates, as our analysis and imputation models do not involve the same set of independent variables [bib_ref] Inference for imputation estimators, Robins [/bib_ref] [bib_ref] Multiple-imputation inferences with uncongenial sources of input, Meng [/bib_ref]. Despite these limitations, the estimate that 57% of all deaths were not recorded in patient record systems is similar to an estimate of 65% in South Africa, which was also found to increase over time, from around 40% of deaths before 2006 to 66% in 2011-2014 [bib_ref] A comparison of death recording by health centres and civil registration in..., Johnson [/bib_ref]. This increase over time in the fraction of deaths that are unrecorded, both in our analysis and the South African study, is a reflection of increasing LTFU in more recent periods [bib_ref] Outcomes of antiretroviral therapy over a 10-year period of expansion: a multicohort..., Grimsrud [/bib_ref].
Estimates of AIDS mortality globally have been contentious and have differed markedly across models [bib_ref] Embracing different approaches to estimating HIV incidence, prevalence and mortality, Hallett [/bib_ref] [bib_ref] Assessment of epidemic projections using recent HIV survey data in South Africa:..., Eaton [/bib_ref]. The substantial heterogeneity in mortality rates shown in our analysis, between and within regions, suggests that more precise and accurate quantification of local ART programme impact will require the reconciliation of mortality data from research cohorts (such as presented in the current analysis) and mortality data from vital registration and case reporting systems. This analysis lays the foundation for an evidence synthesis approach that appropriately weights different mortality data sources when producing model estimates of AIDS mortality at a country level. These estimates are critical for evaluating the success of ART programmes and for identifying the subpopulations most affected by AIDS mortality.
[fig] Figure 1: Cumulative mortality hazard, by region. Sub-Saharan African results are obtained by imputing mortality outcomes in patients who were lost to follow-up. period), a sensitivity analysis was conducted in which additional duration categories were included in the model (12-23 months, 24-35 months and !36 months). In this sensitivity analysis, the decline in mortality over time remained significant, but was not as substantial as in the main analysis, and in the African regions mortality appeared to be higher only in the 2001-2006 period(Fig. 2). The ratio of the mortality rate at 24-35 months to that at 12-23 months was 0.85 (95% CI: 0.75-0.96) in the non-African regions and 0.78 (95% CI: 0.75-0.81) in the African regions, whereas the ratio of the mortality rate at least 36 months to that at 12-23 months was 0.78 (95% CI: 0.70-0.87) in the non-African regions and 0.62 (95% CI: 0.60-0.64) in the African regions [/fig]
[fig] 43: (0.36-0.53) 0.39 (0.31-0.49) 0.44 (0.41-0.48) 0.42 (0.36-0.49) 500þ 0.45 (0.37-0.56) 0.33 (0.25-0.43) 0.36 (0.33-0.40) 0.24 (0.08-0.77) ART, antiretroviral treatment. a In Asia and the Americas, ART eligibility was assessed based on clinical and immunological criteria, depending on the published ART eligibility criteria for the country in the relevant year. In sub-Saharan Africa, ART eligibility was assessed based only on immunological criteria, due to incomplete recording of clinical stage. The results in the 'After exclusions' analysis are the same as those shown in [/fig]
[table] Table 1: Patient characteristics at antiretroviral treatment initiation, 2001-2017. ART, antiretroviral treatment; RSA, Republic of South Africa. [/table]
[table] Table 2: The model was fitted to data from 4751 adult patients traced after LTFU, in whom a vital status could be established. The model controlled for differences in mortality rates between programmes (results not shown). The Weibull scale parameter is calculated as the average of the coefficients across all programmes. ART, antiretroviral treatment; CI, confidence interval; LTFU, lost to follow-up. [/table]
[table] Table 3: Multivariable analysis of mortality after antiretroviral treatment initiation. [/table]
[table] Table 4: Effects of baseline CD4 R cell count (cells/ml) on mortality before and after excluding patients who did not meet antiretroviral treatment eligibility criteria at the time of antiretroviral treatment initiation a . [/table]
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Machine-learning facilitates selection of a novel diagnostic panel of metabolites for the detection of heart failure
the metabolic derangement is common in heart failure with reduced ejection fraction (Hfref). the aim of the study was to check feasibility of the combined approach of untargeted metabolomics and machine learning to create a simple and potentially clinically useful diagnostic panel for Hfref. the study included 67 chronic HFrEF patients (left ventricular ejection fraction-LVEF 24.3 ± 5.9%) and 39 controls without the disease. Fasting serum samples were fingerprinted by liquid chromatographymass spectrometry. Feature selection based on random-forest models fitted to resampled data and followed by linear modelling, resulted in selection of eight metabolites (uric acid, two isomers of Lpc 18:2, LPC 20:1, deoxycholic acid, docosahexaenoic acid and one unknown metabolite), demonstrating their predictive value in Hfref. the accuracy of a model based on metabolites panel was comparable to BNP (0.85 vs 0.82), as verified on the test set. Selected metabolites correlated with clinical, echocardiographic and functional parameters. the combination of two innovative tools (metabolomics and machine-learning methods), both unrestrained by the gaps in the current knowledge, enables identification of a novel diagnostic panel. Its diagnostic value seems to be comparable to BNP. Large scale, multi-center studies using validated targeted methods are crucial to confirm clinical utility of proposed markers.Despite significant improvement in pharmacological and invasive treatment of cardiovascular diseases (CVD) that led to an increase in life expectancy, the incidence of heart failure with reduced ejection fraction (HFrEF) continues to rise. As a result, HFrEF has become a major public health problem as it constitutes currently a leading cause of hospital admissions over the age of 65 1 . In spite of rapidly expanding current knowledge, molecular basis of the HFrEF is still incompletely understood. Available biomarkers, though playing an important role in the HFrEF diagnosis, risk stratification and prognosis (eg.: B-type natriuretic peptide -BNP, N-terminal pro B-type natriuretic peptide -NTproBNP, suppression of tumorigenicity-2 -ST2 family, galectin-3 -Gal-3, troponin -Tn etc.), do not give a satisfactory answer about HFrEF pathophysiology. Moreover there are some significant limitations that may lower clinical utility of natriuretic peptides (NPs: BNP or NTproBNP) which are most established HF biomarkers in clinical practice (e.g. limitation in detection of early and asymptomatic stages of HFrEF, an increase in NPs concentration due to the several cardiac as well as non-cardiac conditions, NPs concentration rising with age, are higher in women, while lower in obese patients). None of currently available HF biomarker do provide additional therapeutic target possibilities nor enable biomarker guided therapy in chronic HFrEF 2 . In fact, an assessment of a single, particular metabolite or protein cannot provide fully informative results. Each of the currently available HFrEF biomarkers being restricted to particular mechanism (e.g. NPs -pressure-volume overload, ST2 family -inflammation, Gal-3 -fibrosis and cardiac remodeling etc.) cannot reflect simultaneously all other mechanisms related to HFrEF pathophysiology. HFrEF being a multisystemic syndrome 3 needs a holistic approach to study systemic changes occurring in the course of HFrEF development. Due to the aforementioned
limitations there is still a need to search of newer HFrEF biomarkers with particular emphasis on a multibiomarker approach. Untargeted metabolomics analysis enables comprehensive characterization of low molecular weight metabolites reflecting the complete metabolic phenotype of the disease. Therefore the importance of this hypothesis free, complex metabolic evaluation has increased within the last years and metabolomics approach has been more frequently used in the search for new HFrEF biomarkers. However, the number of small-molecule metabolites detected by using untargeted metabolomics approach range from hundreds to thousands. As a result, a problem of massive amounts of data has appeared generating a need of specialized forms of data analysis. Studies that have been conducted so far were restricted mainly to classical regression-based models which constitute significant limitation especially in terms of pre-specification of a model structure (based on a theory and assumption), the number of variables included in the analysis and their interactions. To our knowledge, in none of the available HFrEF untargeted metabolomics studies machine-learning (ML) algorithms have been used to analyze metabolomics data. ML, by having different motivating philosophies (data-driven models) and by not being limited by current knowledge (no need for a pre-specification of a model structure), seems to be a powerful tool to improve diagnostic and prognostic processes in various diseases [bib_ref] Diagnosis of heart failure with preserved ejection fraction: machine learning of spatiotemporal..., Tabassian [/bib_ref] [bib_ref] Machine learning for real-time prediction of complications in critical care: a retrospective..., Meyer [/bib_ref] [bib_ref] Heart failure: diagnosis, severity estimation and prediction of adverse events through machine..., Tripoliti [/bib_ref] [bib_ref] Analysis of machine learning techniques for heart failure readmissions, Mortazavi [/bib_ref]. Therefore, the aim of the study was to detect in peripheral blood possibly all low molecular weight metabolites which differentiate HFrEF patients from controls with further creation of the top performing diagnostic panel using ML algorithms.
# Material and methods
Study population. Study population, methodology for obtaining peripheral blood samples, clinical and metabolomics evaluation were as described previously [bib_ref] LC-MS-based serum fingerprinting reveals significant dysregulation of phospholipids in chronic heart failure, Marcinkiewicz-Siemion [/bib_ref]. In brief, 67 patients with chronic heart failure with reduced ejection fraction (HFrEF) and 39 age-, ischemic heart disease (IHD) occurrence-and body mass index (BMI)-matched controls were enrolled in the study. Patients' (HFrEF and control group) inclusion to the study was conducted between 2012-2015 at Cardiology Department of the University Hospital in Bialystok, Poland. The investigation conforms with the principles outlined in the Declaration of Helsinki. The Bioethical Commission of the Medical University of Bialystok approved the research (R-I-002/67/2013). HFrEF study group consisted of ambulatory optimally treated patients (according to the 2012 European Society of Cardiology guidelines for the diagnosis and treatment of acute and chronic heart failure) with stable moderate chronic HFrEF (left ventricular ejection fraction -LVEF ≤ 35%) who did not have an episode of decompensation within the last month. All of the HFrEF patients had a minimum six-month history of the disease. Clinical, biochemical (BNP) and echocardiographic (LVEF ≥ 50%) assessment allowed to exclude HF from the control group which consisted of ambulatory treated patients with arterial hypertension, atrial fibrillation, ischemic heart disease and/or hypercholesterolemia. All of the study participants provided written informed consent. The exclusion criteria were the same for both groups: acute and chronic inflammatory diseases (rheumatoid arthritis and asthma), severe chronic obstructive pulmonary disease (forced expiratory volume in one second -FEV1 less than 50% of a predicted value), severe renal dysfunction (estimated glomerular filtration rate -eGFR < 30 ml/min/1.73 m²), diabetes mellitus, thyroid dysfunction requiring pharmacotherapy, a history of implantation of the cardiac resynchronization therapy device (CRT) or diagnosis of cancer in the past five years. Information about treatment, prior hospitalizations and concomitant diseases were gathered from the medical documentation. Clinical (body mass index -BMI, systolic and diastolic blood pressure -SBP/DBP, heart rate -HR, New York Heart Association -NYHA class), biochemical (complete blood count, iron level, parameters of renal function, urea, uric acid, fasting lipid profile, natriuretic peptide -BNP, C reactive protein -CRP), echocardiographic (left ventricular ejection fraction -LVEF, left ventricular end-diastolic diameter -LVEDd) and functional (six-minute walk test -6MWT, cardiopulmonary exercise test -CPET with rest spirometry) assessment were carried out in all of the enrolled patients. Clinical examination, basic biochemical analyses, echocardiography and functional assessment were done at the day of inclusion to the study. Metabolomics assessment was performed in two batches in 2014 and 2015 after collecting complete sets of biological material within the HFrEF and control group. Each batch included both HFrEF and control group patients. clinical parameters. BMI was calculated as weight [kg]/height [m]². The modification of diet in renal disease (MDRD) formula GFR calculator was used to compute estimated glomerular filtration rate (eGFR). The incidence of ischaemic heart disease (IHD) was calculated based on medical documentation (HFrEF -invasively confirmed IHD as a cause of chronic HF; control group -previous diagnosis of IHD). For cardiopulmonary exercise test (CPET), a standardized continuous Ball State University/Bruce (BSU/Bruce) Ramp treadmill protocol was used with 20-second stages and continuous increase in workload per stage. Forty four (66%) patients with chronic heart failure carried out CPET. The decision about patient's ability to perform the CPET was made after the 6MWT. Difficulties with walking due to non-cardiac causes (e.g. peripheral occlusive arterial disease, problems with lumbar spine), significant fatigue in the 6MWT, fear of exercise on a treadmill or mask intolerance were main reasons of not performing CPET.
Peak oxygen uptake (PVO2) is the rate of an oxygen consumption reflecting the difference between inspired and expired volume of oxygen. PVO2 is measured at peak exercise on a treadmill and expressed as milliliters of O 2 per kg per minute (mL/kg/min). PVO2 depends on the arteriovenous O 2 difference and cardiac output reserve. The slope of a minute ventilation to carbon dioxide output (VE/VCO2 slope) reflects the effectiveness of ventilation. An increase in VE/VCO2 slope in HFrEF patients is known as an indicator of poor outcome.
Metabolic fingerprinting by LC-QTOF-MS. In order to avoid variation due to circadian rhythm, the collection of peripheral venous blood samples was carried out in the morning (at the day of inclusion to the study) between 8.00 and 10.00 am after compulsory overnight fasting (at least 8 h). Samples were further centrifuged for 10 minutes (1300 x g, room temperature). The separated serum was stored in the Eppendorf tubes at −80 °C until further metabolomics analysis. All of the morning medications were taken as usual. Collected serum samples were further subjected to untargeted analysis by liquid chromatography -quadrupole time-of-flight -mass spectrometry (LC-QTOF-MS -model 6550, Agilent Technologies, Santa Clara, California, USA) system. The analytical process was controlled by the use of quality control (QC) samples [bib_ref] Controlling the quality of metabolomics data: new strategies to get the best..., Godzien [/bib_ref]. As the LC-MS analyses were performed in two separate sets (1 st set: 36 HFrEF patients and 19 age-matched controls; 2 nd set: 31 HFrEF patients and 20 age-, gender-and concomitant disease-matched controls), a pool of equal volumes of serum from each of the 55 samples in derivation and 51 in validation sets were used to prepare the QCs. They were prepared independently following the same procedure as for the rest of the samples and injected at the beginning of the run and after every 6-7 real samples. Samples were analyzed by the HPLC system that consisted of a degasser, two binary pumps and thermostated auto sampler connected to a mass spectrometry detector using previously described method [bib_ref] LC-MS-based serum fingerprinting reveals significant dysregulation of phospholipids in chronic heart failure, Marcinkiewicz-Siemion [/bib_ref]. Samples from each set (derivation and validation) were analyzed in a randomized order in separate runs (first for positive and then for negative ion mode). Metabolomics analyses were conducted at Clinical Research Centre of Medical University of Bialystok, Poland.
Processing of LC-MS data was performed as described previously [bib_ref] LC-MS-based serum fingerprinting reveals significant dysregulation of phospholipids in chronic heart failure, Marcinkiewicz-Siemion [/bib_ref]. Briefly, the raw data collected by the analytical instrumentation was cleaned of background noise and unrelated ions by the Molecular Feature Extraction (MFE) tool in the Mass Hunter Qualitative Analysis Software (B.06.00, Agilent). Alignment, quality assurance 9 and data filtering were performed using Mass Profiler Professional (MPP) 12.6.1 (Agilent).
# Statistical analysis.
Basic statistical analyses were performed as described previously [bib_ref] LC-MS-based serum fingerprinting reveals significant dysregulation of phospholipids in chronic heart failure, Marcinkiewicz-Siemion [/bib_ref]. In short, continuous variables are expressed as the mean ± standard deviation (SD) or median and interquartile range (IQR), depending on the type of distribution. Categorical variables are presented as raw values and percentages from the total. The Student's t-test, Mann-Whitney U test or χ² test were used, as appropriate. Statistical significance was defined as p < 0.05.
As the data were obtained by subjecting samples to MS analysis in two separate batches, which imposed further manual matching, the variables were prefiltered to include 50% most variant metabolites of these present in at least 80% of samples. Following the filtering, manual matching and elimination of artifacts (signals present in blank analysis), 63 variables from both ESI ion modes have been subjected to computational modelling. Missing values were replaced by k-means nearest neighbour analysis according to the criteria of Armitage et al. [bib_ref] Missing value imputation strategies for metabolomics data, Armitage [/bib_ref].
There were nine cases with missing BNP, which were omitted in models. No data imputation techniques were applied, since gathered data would not allow us to obtain a reliable approximation of unobserved BNP levels. Within each batch, the variables were transformed with Yeo-Johnson method and standardized by subtracting the mean and dividing by trimmed standard deviation. Cases were split into training and test set, with proportions of 3:2. Recursive feature elimination with repeated cross-validation (RFECV) based on random forests was performed on the training set to rank the predictors. Top 20 ranking metabolites were then subset and incrementally used as predictors of multiple GLMs fitted to training set, with their accuracy assessed on resampling. The top performing model, containing 8 predictors, was chosen and its validity was assessed on the test set. Additional multiple regression models, including HFrEF presence and another clinical covariate as predictors, one for each clinical covariate and metabolite combination, have been built to assess potential confounding on metabolite levels. Correlations between serum intensities of metabolites included in the panel and clinical parameters were performed calculating Pearson's product-moment coefficients for pairs of normalized variables. The analysis was performed in R version 3.4.2.
Metabolites identification. Identification of significant metabolites was performed as described previously [bib_ref] LC-MS-based serum fingerprinting reveals significant dysregulation of phospholipids in chronic heart failure, Marcinkiewicz-Siemion [/bib_ref]. Identification of uric acid, deoxycholic acid and docosahexaenoic acid was confirmed by matching retention time, accurate mass and fragmentation pattern of authentic standards (Sigma Aldrich). Lysophospholipids were identified based on previously described fragmentation pattern 9 . . There were no statistically significant differences between study groups in terms of age, blood pressure, BMI and IHD occurrence. The percent of women was higher within the control group (n = 11, 29% vs n = 7, 10%, respectively; p = 0.019). HFrEF group consisted of mildly/moderately symptomatic patients (NYHA class II -43%; III − 57%) who had significantly impaired left ventricular ejection fraction (EF 24.3 ± 5.9%). Ischemic heart disease was an etiology of HFrEF in 57% of patients. HFrEF patients presented slower resting heart rate than controls (70.7 ± 9.9 vs 75.3 ± 12.5 beats per minute, p = 0.045). Among assessed laboratory parameters higher concentration of uric acid (6.8 mg/dL IQR 6.0-7.9 mg/dL vs 5.99 mg/dL IQR 5. . Basic clinical characteristics of patients included in the study (chronic heart failure and controls without the disease). ¹Median (IQR); ²Mean (SD); IQR -interquartile range; SD -standard deviation; *CPET was performed in 66% (n = 44) of HFrEF patients; ∫ IHD -invasively confirmed ischaemic heart disease as the etiology of heart failure; **IHD -invasively confirmed or clinically diagnosed and pharmacologically treated ischaemic heart disease; HFrEF -heart failure with reduced ejection fraction; NYHA class -New York Heart Association functional classification; SBP/DBP -systolic/diastolic blood pressure; MAP -mean arterial pressure; HR -heart rate; BMI -body mass index; RBC -red blood cells; Hb -haemoglobin; Fe -serum iron level; CRP -C-reactive protein; eGFR -estimated glomerular filtration rate; TChol -total cholesterol; LDL -low-density lipoproteins; HDL -high-density lipoproteins; TG -triglycerides; BNP -B-type natriuretic peptide; LVEF -left ventricular ejection fraction; LVEDd -left ventricle end-diastolic diameter; 6MWT -sixminute walk test; CPET duration -cardiopulmonary exercise test duration; Peak VO2 -peak rate of oxygen uptake; VE/VCO2 slope -minute ventilation/carbon dioxide production slope; AF -atrial fibrillation; CKD -chronic kidney disease (eGFR <90 ml/min/1.73 m²); ASA -acetylsalicylic acid; ACEIs/ARBs -angiotensinconverting-enzyme inhibitors/angiotensin II receptor blockers; MRAs -mineralocorticoid receptor antagonists; CCBs -calcium channel blockers. www.nature.com/scientificreports www.nature.com/scientificreports/ Selection of top ranked metabolites which created the diagnostic panel. Quality assurance protocol, manual matching and filtering of further artifacts were carried out on the data obtained from the two independent LC-QTOF-MS analyses. As a result, 63 variables have been subjected to computational modelling. Random forests nested inside RFECV algorithm lead to selection of 20 top ranked predictors, further sequentially used as covariates of GLMs, of which the most accurate final model was chosen, built on 8 metabolites [fig_ref] Equation 1 y: Model equation, for the logit-link binomial model, relating HFrEF occurrence [/fig_ref] , . Further MS/MS spectrum analyses enabled to identify components of the model (uric acid, two isomers of lysophosphatidylcholine -LPC 18:2, LPC 20:1, deoxycholic acid, docosahexaenoic acid and one unknown metabolite), and build the final model equation [fig_ref] Equation 1 y: Model equation, for the logit-link binomial model, relating HFrEF occurrence [/fig_ref]. Apart from uric acid serum intensity of seven remaining metabolites was significantly lower in HFrEF . Based on both accuracy profiling [fig_ref] Figure 2: Accuracy [/fig_ref] , and ROC curve [fig_ref] Figure 3: Receiver operating curve [/fig_ref] , a prediction cut-off value equal to 0.5 was found to be optimally discriminate between controls and HFrEF cases. Our model compared to BNP as a sole predictor presents with better accuracy (0.85 vs 0.82; [fig_ref] Equation 1 y: Model equation, for the logit-link binomial model, relating HFrEF occurrence [/fig_ref] and specificity (0.92 vs 0.83), but worse sensitivity (0.73 vs 0.80). Overall performance measured by AUC is insignificantly lower (0.85 vs 0.92, p = 0.29). To demonstrate the additive value of metabolite panel on top of BNP, we have built an additional model containing all these predictors (Supplementary . Formal testing confirmed better goodness-of-fit of full model than BNP alone (p = 0.001), while resampling profile indicates comparable accuracy [fig_ref] Figure 4: Comparison of model accuracies estimated on 25 runs of data resampling [/fig_ref]. Additional multiple linear regression models have been built, to investigate the potential effect of other clinically relevant covariates (such as ischaemic ethiology, statin or ACEI treatment) on metabolite levels [fig_ref] Figure 2: Accuracy [/fig_ref]. Although in some cases these have shown some extent of potential confounding, they did not nullify the initial relationship with HFrEF.
Serum intensities of all metabolites significantly correlated with left ventricular ejection fraction. Moreover, apart from UA and DHA, serum intensities of remaining metabolites moderately positively correlated with exercise duration on a treadmill, peak oxygen consumption, renal function and negatively correlated with VE/VCO2 slope. Serum intensities of lysophosphatidylcholines (LPCs) included in the metabolites' panel correlated positively with LDL and total cholesterol serum level. Weak negative correlation was also observed between deoxycholic acid, LPC 18:2 sn1 and BNP .
# Discussion
As heart failure is not a single organ disease but a multisystemic syndrome, variety of systemic changes occur in the course of HFrEF development. Analysis of changes in blood metabolites profile seems to be an attractive approach to perform holistic assessment of complex adaptive responses and to discover valuable novel HFrEF biomarkers. An untargeted metabolomics analysis being unrestricted by current knowledge enables detection of possibly all low molecular weight metabolites present in a particular moment in the peripheral blood. That allows a simultaneous assessment of changes in many various metabolites, including even those that we have not been able to identify to date. Our study presents a completely new approach towards biomarkers -something to be expected in the era of personalized medicine when "one fits all" approach will be replaced by analysis of simultaneous changes of multiple substances reflecting different mechanisms. According to current research, collective biomarkers better reflect the complexity of changes in the organism and possibly will increase likelihood to define particular sub-phenotypes of diseases (in this case heart failure) [bib_ref] Emerging role of precision medicine in cardiovascular disease, Leopold [/bib_ref] [bib_ref] Novel endotypes in heart failure: effects on guideline-directed medical therapy, Tromp [/bib_ref] [bib_ref] The continuous heart failure spectrum: moving beyond an ejection fraction classification, Triposkiadis [/bib_ref]. Previous research has already proven that metabolomics may constitute a powerful diagnostic and prognostic tool in chronic HF [bib_ref] Metabolic disturbances identified in plasma are associated with outcomes in patients with..., Cheng [/bib_ref] [bib_ref] Prognostic value of elevated levels of intestinal microbe-generated metabolite trimethylamine-N-oxide in patients..., Tang [/bib_ref]. However, a high diversity of analytical methods (e.g. nuclear magnetic resonance -NMR, mass spectrometry -MS), separation techniques (i.e. capillary electrophoresis or chromatography -gas or liquid) and approaches (untargeted, targeted) used to date to study metabolome as well as various studied populations are partly responsible for a diversity in the results obtained by different research groups. For instance, Cheng et al. [bib_ref] Metabolic disturbances identified in plasma are associated with outcomes in patients with..., Cheng [/bib_ref] have performed untargeted metabolomics analysis followed by targeted evaluation of obtained results and an identification of a combination of four metabolites (histidine, phenylalanine, spermidine, and phosphatidylcholine C34:4) that discriminated HF stage C from control group similarly to b-type natriuretic peptide (BNP). Authors have suggested that profile of . Mean serum intensities of top ranked metabolites included in the panel in chronic heart failure versus control group (without partitioning into training and test set). RT -retention time; m/z -mass-to-charge ratio; HFrEF -heart failure with reduced ejection fraction; SD -standard deviation; UA -uric acid; LPClysophosphatidylcholine; UM -unknown metabolite; DA -deoxycholic acid; DHA -docosahexaenoic acid. [bib_ref] Metabolomic profiling identifies novel circulating biomarkers of mitochondrial dysfunction differentially elevated in..., Hunter [/bib_ref] included healthy control group. As the metabolites' profile is susceptible to many various external (e.g. pharmacotherapy) and internal (e.g. age, sex, comorbidities, renal function) factors, many differences in basic characteristic of HF and control group (e.g. age, BMI, comorbidities, renal function, pharmacotherapy) observed in those studies may serve as significant confounding factors. On the contrary to previous research, control group in our study was carefully matched in terms of age, BMI, eGFR, IHD occurrence. Despite this, differences in medication were noticed. Additional analyses showed the relation between medications and serum intensities of UA (ACEI treatment), LPC 18:2sn1 (ACEI, statin therapy) and HFrEF [fig_ref] Figure 3: Receiver operating curve [/fig_ref]. However, due to small group size these analyses are biased by strong association between the treatment and the presence of heart failure. Previous metabolomics studies 14 that used untargeted analysis to select metabolites and further create a novel diagnostic panel for HFrEF have been based on classical, hypothetical-driven statistical approaches. In fact, the amount of data gathered from an untargeted metabolomics analyses poses an analytical challenge resulting from a necessity of modelling a multidimensional space of relations between metabolite fingerprint and outcomes, and simplifying the results just enough to facilitate holistic, straightforward conclusions. An application of machine-learning techniques efficiently solves these tasks by incorporating multiple procedures consisting of data resampling and remodelling. In our case, this eventually allows to acquire a single, simple linear model, that reapplied to patients' metabolic data yields a value, that might be interpreted as a 'surrogate metabolite' , being a linear combination of set of selected metabolites, which might be used for discrimination of heart failure cases, after applying a single cut-off. To our knowledge, this is the first study that implemented untargeted metabolomics analysis combined with ML algorithms in order to select metabolites creating the top performing diagnostic model for HFrEF. Recently, Verdonschot et al. 18 www.nature.com/scientificreports www.nature.com/scientificreports/ have shown that the NT pro-BNP-based determination of the dilated cardiomyopathy (DCM) severity might be complemented by the combination of metabolites. In contrast to our study, authors have applied targeted metabolomics approach which might limit objective analysis of changes occurring in the blood/urine metabolites' profile. The use of non-fasting blood samples, significant differences in diabetes prevalence within the studied groups, lack of chronic inflammatory disease exclusion could potentially influence the disease metabolic phenotype. Nevertheless, in spite of these study limitations the result of the analysis suggest some clinical utility of the combination of metabolomics and ML methods. In our study an untargeted metabolomics analysis was followed by computational modelling which enabled hypothesis free selection of a panel of metabolites which seemed to have non-inferior diagnostic value (based on accuracy) to BNP in chronic HFrEF. Additionally, the results of the likelihood-ratio test performed for GLMs suggested that prediction of the outcome with model utilizing BNP and eight metabolites presents significantly higher goodness-of-fit when compared with BNP alone. When we based our conclusions on data resampling, our fit slightly outperformed the BNP alone, however for such a small set, this clearly remains inconclusive.
According to the previous research, all of the selected and identified metabolites have already been described in various cardiovascular diseases (CVD) including heart failure. However, apart from DHA (possible adjunctive therapy in optimally treated patients with symptomatic HFrEF) and uric acid (useful marker of adverse prognosis in HFrEF patients), clinical significance of changes in residual components of a panel in HFrEF remains unknown. In our study HFrEF patients presented significantly higher serum UA level in comparison with the control group. According to the literature, the incidence of hyperuricemia is high in HFrEF and occurs in 50-55% of patients. An elevation in uric acid level is considered to be a result of increased production via higher xanthine oxidase activity or decreased elimination via kidneys. In our study, both groups (controls and HFrEF) were fitted in terms of estimated glomerular filtration rate (eGFR) and patients with severe chronic kidney disease were excluded, reducing the role of UA excretory disorders. It is still unclear why UA itself has a negative impact on prognosis in HFrEF 19,20 as randomized trials with agents blocking this pathway failed to provide clinical benefit [bib_ref] OPT-CHF Investigators. Impact of oxypurinol in patients with symptomatic heart failure. Results..., Hare [/bib_ref]. Hypothesis free selection of UA as a component of a top performing diagnostic panel confirms the need for further research and continuation of a discussion on xanthine oxidase metabolic pathway (including serum uric acid -sUA) role in HFrEF pathophysiology. Lipid disorders have also been recognized as a strong risk factor for CVD. Nevertheless, the vast majority of previous research regarding dyslipidemia in chronic HFrEF concerned solely alteration in cholesterol metabolism. As a result of a growing interest in the matter of metabolomics and lipidomics within the last years, it was possible to detect a substantial dysregulation in metabolism of other lipid fractions, including phospholipids [bib_ref] Controlling the quality of metabolomics data: new strategies to get the best..., Godzien [/bib_ref] [bib_ref] Metabolic disturbances identified in plasma are associated with outcomes in patients with..., Cheng [/bib_ref] [bib_ref] A novel lipid biomarker panel for the detection of heart failure with..., Mueller-Hennessen [/bib_ref]. This suggests more complex lipid metabolism abnormalities in HFrEF. In this study, serum intensities of all LPCs, likewise cholesterol level, were significantly lower in the HFrEF group. Simultaneously, higher percentage of statin use was observed in HFrEF patients. According to the results of additional analysis, it seems that statin therapy was likely to partialy impact the association between LPCs (e.i. LPC 18:2sn1) and HFrEF. Therefore, the differences between both groups (HFrEF and controls) regarding statin therapy might be considered as a possible study limitation. Although, an assessment of each particular metabolite is a kind of simplification as panel compounds should be considered together as a "surrogate metabolite". In our previous study it has been shown that the greater serum PLs deficit, the worse clinical condition of HF patient (including more severe metabolic dysregulation, impaired renal function and decreased exercise capacity) [bib_ref] Controlling the quality of metabolomics data: new strategies to get the best..., Godzien [/bib_ref]. Despite the fact that an alteration in PLs metabolism is considered to be related to plethora of processes associated with HFrEF (e.g. immune response, impaired energy metabolism, altered choline metabolism with a possible role of gut microbiota), the exact metabolic mechanism responsible for changes in PLs in HFrEF remains unknown. The dysregulation in phospholipids including phosphatidylcholines (PCs) metabolism has already been observed in various non-related diseases 9,22-25 . Lindahl et al. [bib_ref] Overlap in serum metabolic profiles between non-related diseases: implications for LC-MS metabolomics..., Lindahl [/bib_ref] suggested that alteration in LPCs concentration may be an indicative of disease in general rather than a disease specific metabolite marker. In HFrEF which is a multisystemic syndrome, changes observed in the LPCs serum intensities considered as a part of the whole metabolites' profile seem not to be a limitation but an advantage. The presence of LPCs in the diagnostic panel and their correlations with serum cholesterol level points out an importance of dysregulation in various lipid classes in HFrEF. Bile acids (BAs), likewise LPCs and UA, have been described as factors implicated in various cardiac pathologies. Mayerhofer et al. [bib_ref] Increased secondary/primary bile acid-ratio in chronic heart failure, Mayerhofer [/bib_ref] have demonstrated that the ratio of primary to secondary BAs has been reduced in chronic heart failure patients. Nevertheless, the association between this pattern of BAs composition and reduced overall survival has been seen solely in univariate analysis. In our study, serum intensity of one secondary BA (deoxycholic acid -DCA) was lower in HFrEF group. Deoxycholic acid is known as one of the two most common secondary bile acids that are synthesized solely by the microbial flora of a small intestine. An impairment in intestinal function and gut microbiome in HF has been intensively studied within the last years [bib_ref] The importance of the gastrointestinal system in the pathogenesis of heart failure, Krack [/bib_ref] [bib_ref] Novel concept of heart-gut axis in the pathophysiology of heart failure, Kamo [/bib_ref]. Reduced intestinal blood flow with further bowel wall oedema has been thought to be responsible for altered intestinal barrier function leading to the passage of bacterial products into the systemic blood circulation. Another component of a diagnostic panel with known anti-inflammatory, anti-arrhythmic and beneficial effects on the endothelial function was docosahexaenoic acid (DHA) classified as omega-3 polyunsaturated fatty acid (PUFA) [bib_ref] Effects of n-3 polyunsaturated fatty acids on left ventricular function and functional..., Nodari [/bib_ref] [bib_ref] Low levels of cellular omega-3 increase the risk of ventricular fibrillation during..., Aarsetoy [/bib_ref]. Mozaffarian et al. [bib_ref] Circulating long-chain omega-3 fatty acids and incidence of congestive heart failure in..., Mozaffarian [/bib_ref] have indicated that circulating omega-3 PUFAs are associated with lower incidence of chronic heart failure. In our study serum intensity of DHA was significantly lower than in the control group. Current European Society of Cardiology (ESC) guidelines for the diagnosis and treatment of acute and chronic heart failure has recommended that n-3 PUFA preparations containing >850 mg/g of eicosapentaenoic acid (EPA) and DHA may be considered as an adjunctive therapy in optimally treated patients with symptomatic HFrEF 1 . Endothelial dysfunction, oxidative stress, systemic inflammatory activation, impaired energy metabolism, altered choline metabolism, apoptosis, intestinal dysbiosis -all of those have been thought to be implicated in HFrEF pathophysiology. As cited above, every metabolite included in the diagnostic panel has already been described to be involved in an activation of aforementioned processes. Moreover their correlations with clinical, biochemical (BNP, renal function), echocardiographic (left ventricular systolic function) and functional parameters (the distance of 6MWT, duration of exercise on a treadmill, peak VO2, VE/VCO2 slope) confirm possible clinical significance of the diagnostic panel components. Therefore, based on the results of this and previous studies, metabolomics seems to be a powerful tool in HFrEF especially when combined with ML algorithms in the detection of the top performing HFrEF diagnostic panel. Presence of additional unidentified metabolite in the panel only confirms great possibilities that the combination of those two unrestricted methods offers in the aspect of broadening the knowledge on HFrEF pathophysiology. As compared to the strategy of a single metabolite, complementarity of a panel compounds gives an opportunity to get a more complete picture of the metabolic changes taking place in the course of HFrEF and as a result may increase accuracy of the diagnostic panel.
Study limitations. Despite the study was carefully planned we are aware that there are several potential limitations. First, number of enrolled patients is relatively small. Nevertheless, our purpose was to select the most homologous HFrEF group using strict exclusion criteria especially in terms of concomitant diseases. Second, there is no information about patients' diet, PUFAs supplementation or HFrEF duration. Third, there are differences in pharmacotherapy between study and control group. Larger studies with prospectively followed-up groups are needed for clinical validation of the diagnostic panel.
# Conclusions
In the present study we demonstrated that the combination of two innovative methods: an untargeted metabolomics and ML algorithms can be a promising tool for the diagnostic workup in HFrEF. The combination of metabolites may provide comparable diagnostic value to BNP. Due to the complementarity of the panel components, changes in the serum intensities of particular metabolites interpreted together as a "surrogate metabolite" might be more specific to the HFrEF. Large scale, multi-center studies using validated targeted methods are crucial to confirm clinical utility of proposed markers.
[fig] Equation 1 y: Model equation, for the logit-link binomial model, relating HFrEF occurrence (outcome) to metabolite panel (predictors). Predictors are 0-centered, Yeo-Johnson normalized intensities of metabolites. = 1.781 + −3.190 * [LPC 18:2sn2] + 0.851 * [UA] + 0.037 * [LPC 18:2sn1] + −0.035 * [UM] + 2.749 * [LPC 18:2sn2] + −1.099 * [LPC 20:1] + −0.673 * [DA] + −1.099 * [DHA]. [/fig]
[fig] Figure 1: Accuracy (%) for metabolites' panel (in relation to the number of predictors included in the panel) in comparison with the accuracy for BNP while assuming a cut-off point equal 35 pg/dl (as for the diagnosis of heart failure of non-acute onset). [/fig]
[fig] Figure 2: Accuracy (%) for metabolites' panel in relation to the single cut-off allowing discrimination of HFrEF cases. Scientific RepoRtS | (2020) 10:130 | https://doi.org/10.1038/s41598-019-56889-8 www.nature.com/scientificreports www.nature.com/scientificreports/ metabolites (another panel identified by Cheng et al.) may provide even better prognostic value in comparison with conventional biomarkers such as BNP in HF patients. Mueller-Hennessen et al.16 have created a lipid diagnostic panel which improved HFrEF detection, even in mild and asymptomatic stages. Hunter et al.17 have indicated a group of circulating metabolites which were significantly elevated in HF in comparison with the control group. Moreover it allowed to differentiate HFrEF from heart failure with preserved ejection fraction (HFpEF).Different analytical approaches were used in all of those studies (Cheng et al. -untargeted followed by targeted analysis; Mueller-Hennessen et al. -metabolite profiling; Hunter et al. -targeted analysis). Apart from this, analyses carried out by Cheng et al. 14 and Hunter et al. [/fig]
[fig] Figure 3: Receiver operating curve (ROC) for eight metabolites' panel and BNP (sensitivity 0.73 vs 0.80, specificity 0.92 vs 0.83; AUC 0.85 vs 0.92, respectively; p = 0.29). [/fig]
[fig] Figure 4: Comparison of model accuracies estimated on 25 runs of data resampling. (2020) 10:130 | https://doi.org/10.1038/s41598-019-56889-8 [/fig]
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Selective loss of function variants in IL6ST cause Hyper-IgE syndrome with distinct impairments of T-cell phenotype and function
## Supplementary data
# Methods
## Exome sequencing and data analysis
Whole exome sequencing was performed using the TrueSeq Rapid Exome kit, Illumina HiSeq3000 system and the cBot cluster generator. Burrows-Wheeler Aligner was used to align reads against version 19 of the human genome as a reference. VCF.Filter tool 1 was used to filter out variants (annotated by SNPEFF) with a minor allele frequency (MAF) >0.01 in 1000 Genomes, ExAC or dbSNP. An internal database was also used to filter out recurrent variants. In the resulting list of variants homozygous changes were then subjected to prioritization using the combined annotation dependent depletion (CADD) tool 2 to predict deleteriousness.
## Sanger sequencing
Validation and segregation of the GP130 variant P498L was performed on DNA from P P498L and family members by Sanger sequencing. DNA from the deceased sister and twin brothers was not available. The following primers were designed for amplification and sequencing: Forward: 5'-CCGAACAGTAGGTCCTTTGG-3', Reverse: 5'-CCACTGCATTGGCAATACTT-3'.
## Patient-derived cells and ko cell line
T Lymphoblasts: T Lymphoblasts were generated by feeder cell-mediated T cell expansion. Irradiated feeder cells (PBMCs and JY cells) were added in a 1:1 ratio to patient PMBCs in T cell media (RPMI 1640 (Gibco) + 5% human serum + 1% penicillin/streptomycin + 1% Hepes) supplemented with IL-2 (100U, ImmunoTools) and PHA (1μg/ml; Sigma). T cells were allowed to expand for two weeks.
Epstein-Barr virus transformed lymphoblastoid cells (EBV-LCL): PBMCs freshly isolated from the patient, mother and healthy donor blood were incubated with EBV virus supernatant for one day followed by addition of 1 µg/ml cyclosporin A. Cells were grown in RPMI 1640 medium (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (HI-FBS), 1% L-glutamine and 1% penicillin/streptomycin (all from Invitrogen).
CRISPR/Cas9 GP130 KO HEK293 cell line: The GP130 KO HEK293 cell line was established in house as described previously [bib_ref] A biallelic mutation in IL6ST encoding the GP130 co-receptor causes immunodeficiency and..., Schwerd [/bib_ref]. Cells were grown in DMEM with glutamine (Sigma) supplemented with 10% FCS (Sigma), 1% sodium-pyruvate (Gibco), 1% non-essential amino acids (Gibco), 1% penicillin/streptomycin (Gibco).
## Plasmid and lentivirus production for reconstitution experiments
pcDNA3.1(+) vector coding for wildtype (WT) or mutant GP130, and wildtype IL-6RA and IL-11RA were purchased from GenScript and expanded in Stbl3 E. coli (Thermo Fisher) according to standard protocols. Plasmids were purified using the EndoFree Plasmid Maxi Kit (Qiagen) or E.Z.N.A.® Endo-Free Plasmid Maxi Kit (Omega bio-tek).
For ectopic expression of WT IL6ST in patient-derived fibroblasts, infectious lentiviral particles were generated as described previously 3 with some adaptations. pLENTI7.3V5-DEST (Geneart; Thermo Fisher) carrying WT IL6ST was co-transfected into HEK293 cells using Lipofectamine2000 (Invitrogen) and the ViraPower lentiviral packaging mix (Invitrogen) according to the manufactures' instructions.
HEK293 cell supernatants were filtered by passing through a 0.45μm filter and added to primary fibroblasts in 6-well plate cultures. Transduction efficiency was ∼70% based on EmGFP expression.
After 24 hours incubation cells were serum starved for 2 hours, followed by cytokine stimulation and p-STAT staining, as described in the Methods section of the article.
## Transcription factor profiling
The expression of T cell lineage-specifying transcription factors was analyzed following surface and intracellular co-staining of 0.5-1x10 6 total PBMCs. Cells were stained for surface markers and for the exclusion of dead cells as described below, fixed and permeabilized using the transcription factor staining buffer set (eBioscience), followed by flow cytometry analysis. The expression of transcription factors was detected with the following antibodies: FOXP3, GATA3, RORγt and TBET.
## Tsne-clustering
t-Distributed Stochastic Neighbor Embedding (TSNE)-based analysis was performed on FCS files compensated for spillover between channels and gated on live CD3 + CD4 + CD8or live CD3 + CD8 + CD4single cells and randomly down-sampled to 10.000 (CD4 + ) or 3000 (CD8 + ) cells per sample. Individual FCS files were then concatenated to generate a single FCS file and subjected to tSNE unsupervised analysis using the FlowJo (Treestar) tSNE plugin 4,5 . The settings were: Iterations = 1000; Perplexity = 200; Eta = 20; Theta = 0.5. Clustering was performed on the following parameters: CD45RA, CD25, CD127, CCR4, CCR6, CCR7, CCR9, CCR10, CXCR3, CXCR5 and CRTh2. All patient T cell analysis were compared to matched 12-year old healthy donors samples (p.P498L) or 7-year old healthy donor samples (p.N404Y) that were stained and acquired on the same day.
# Statistical analysis
In cases when three or more independent experiments were performed, an unpaired two-tailed student t-test or Mann-Whitney-test were performed on Prism (version 5 or 7; GraphPad software, Inc.).
## Protein sequence alignment
Clustal Omega 6 was used to align multiple sequences from different species: NP002175 (Human), NP_001252920 (Macaque), NP_034690 (Mouse), NP_990202 (Chicken) and NP_001106976. . List of Antibodies used for p-STAT assays, GP130 expression, extracellular surface staining, intracellular cytokine staining (ICCS), transcription factor (TF) and chemokine receptor (CCR) profiling. Profiling of CD4 + and CD8 + T cells chemokine receptor expression. Bar graph summary of (A and B) CD4 + and (C and D) CD8 + memory T cells chemokine receptor surface expression shown as frequency of live CD3 + CD4 + CD25or CD3 + CD8 + CD25 -T cells, respectively. Mean + SEM; HD agematched controls (9-14 years): n = 10-11, HD age-matched controls (6-7 years): n = 6-9, P P498L : n = 2-5 independent replicates from PBMCs isolated at 3 distinct time points and at 7 and 4-month distance, P N404Y : n = 3 replicates from 2 independent experiments and PBMCs taken at 5-month distance. Mann-Whitney test; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Some healthy donor control data shown was previously published 5 . .
## Supplementary
## Antibody-fluorophore
Phenotypic characterization of CD8 + CCR6 + memory T cells. (A) Summary of frequencies of TBET + and RORγt + cells as assessed by intracellular staining and expressed as frequencies of CD8 + memory T cells. Mean + SEM; HD (adult): n = 28, HD age-matched controls (9-14 years): n = 8, HD age-matched controls (6-7 years): n = 7, P P498L : n = 4 replicates from two independent experiments from PBMCs isolated at two distinct time points and at 7-month distance, P N404Y : n = 3 replicates from two independent experiments and PBMCs taken at 5-month distance. (B) Dot-plot showing the expression of CCR6 and RORγt in CD8 + memory T cells. (C) Dot-plot presentation illustrating the expression of TBET and RORγt in CD8 + memory T cells. Gates were set using naïve CD8 + T cells as internal negative control.
## Figure s5.
Evaluation of regulatory T cell frequencies.
(A) Bar graph summary of CD25 + FOXP3 + T cell frequencies gated on live CD3 + CD4 + cells: mean + SD: HD (adult): n = 9, HD age-matched controls (9-14 years): n = 7, HD age-matched controls (6-7 years): n = 6, P P498L : n = 3 independent replicates from PBMCs isolated at two distinct time points and at 7-month distance, P N404Y : n = 3 replicates from two independent experiments and PBMCs taken at 5-month distance. (B) Dot-plot presentation of CD25 + FOXP3 + -gated T cell populations according to A. Bar graph summary showing percentages of cells expressing TBET, GATA3 or RORγt within CCRenriched Th-cell subsets and CD3 + CD4 + CD25naïve T cells: mean + SD: HD: n = 23, P P498L : n = 5 independent replicates from PBMCs isolated at two distinct time points and at 7 and 4-month distance, P N404Y : n = 3 replicates from 2 independent experiments and PBMCs taken at 5-month distance. Mann-Whitney test; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
[fig] Figure S1: Functional assessment of STAT family members' phosphorylation in GP130 P498L T lymphoblasts.Bar graph summary of STAT1, STAT2, STAT3, STAT4, STAT5 and STAT6 phosphorylation following stimulation with 100 ng/ml (A) IL-6, (B) IL-27, (C) IL-4, (D) IL-12, (E) IL-21 or (F) IFNβin T lymphoblasts from P P498L and two HDs. Data shown are from 2-3 independent experiments with 2 replicates each. t-test; *p<0.05, **p<0.01, ***p<0.001. [/fig]
[fig] Figure S2: Functional assessment of GP130 P498L fibroblast p-STAT1 response. (A) Dose-escalation curves showing relative mean fluorescence intensity (rMFI) of p-STAT1 after stimulation of P P498L and HD fibroblasts as well as P P498L fibroblasts transduced with wild-type (WT) GP130 with IL-6, IL-11, IL-27, LIF or OSM. Bar graphs (right) showing rMFI of fibroblasts upon stimulation with the highest concentration of the corresponding cytokine (3 (HD), 4 (p.P498L) and 2 (p.P498L + WT GP130) replicates of 2 independent experiments). (B) Dot-plot presentation of p-STAT1 and p-STAT3 costained fibroblasts according to (A) following stimulation with 100 ng/ml IL-6, IL-11, IL-27, LIF or OSM. [/fig]
[fig] Figure S3: Figure S3. [/fig]
[fig] Figure S6: Gating strategy for the analysis of chemokine receptor expression and transcription factor expression in CD4 + T cells.Dot plot examples are shown from one adult healthy donor. [/fig]
[fig] Figure S7: Figure S7. [/fig]
[table] 57 Table S3: Table S2. NIH-HIES score calculated on the basis of the clinical manifestations of P P498L . Summary of clinical manifestations present in P P498L and P N404Y . [/table]
[table] Table S4: Homozygous, non-synonymous rare variants (minor allele frequency (MAF) <0.01 using the ExAC database) found in subject P P498L . CADD algorithm was used to predict functional damage of variants. [/table]
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Cytogenetic markers of susceptibility: influence of polymorphic carcinogen-metabolizing enzymes.
Polymorphisms of xenobiotic-metabolizing enzymes, responsible for individual differences in metabolic activation and detoxification reactions, may profoundly modulate the effects of chemical carcinogens. In the case of genotoxic carcinogens, differences in biological effects due to genetic polymorphisms can be evaluated by cytogenetic methods such as the analysis of chromosomal aberrations (CAs), sister chromatid exchanges (SCEs), micronuclei (MN), and changes in chromosome number. These techniques can be applied to any exposure known to induce such alterations, without additional method development for each exposing agent. The influence of polymorphic genes on the cytogenetic effects of a carcinogen can quickly be tested in vitro using metabolically competent cells collected from donors representing different genotypes or phenotypes. For instance, erythrocytes from individuals positive for glutathione S-transferase Ti (GST1I) express GSTT1, whereas GSTT1-null donors, having a homozygous deletion of the GSTT1 gene, completely lack this detoxification enzyme. This deficiency results in highly increased sensitivity to SCE induction in whole-blood lymphocyte cultures by 1,2:3,4-diepoxybutane, a reactive metabolite of 1,3-butadiene. The same cytogenetic techniques can also be applied as effect biomarkers in studies of human populations exposed to genotoxic carcinogens. For example, elevated rates of chromosome damage have been detected among smokers lacking glutathione S-transferase Ml (GSTM1-null genotype), and the baseline level of SCEs seems to be increased in GSTT1-null individuals. Information obtained from cytogenetic studies of genetic polymorphisms can be used, for example, to recognize the genotoxically relevant substrates of the polymorphic enzymes, to identify genotypes that are susceptible to these genotoxins, to improve in vitro genotoxicity tests utilizing human cells, to increase the sensitivity of cytogenetic endpoints as biomarkers of genotoxic effects in humans, and to direct mechanistic studies and cancer epidemiology. Environ Health Perspect 1 05(Suppl 4): 829-835 (1997)
# Introduction
Susceptibility to the hazardous action of result in chromosome instability syndromes chemicals may derive from genetic or characterized by defects of DNA repair or acquired characteristics of the individual. In recombination, increased baseline or the case of genotoxic carcinogens, suscepti-induced chromosome damage, and high bility can be associated with individual difcancer incidence-may also increase cancer ferences, e.g., in the efficiency of carcinogen in heterozygotes [bib_ref] Hyper-recombination and Bloom's syndrome: microbes again provide clues about cancer, Rothstein [/bib_ref]. Besides such hetmetabolism or in the ability to repair DNA erozygotes, sensitivity to genotoxic carcinolesions induced by the carcinogen. Certain gens could be associated, for example, with recessive genes, which among homozygotes subtle genetic polymorphisms influencing This paper was prepared as background for the Workshop on Susceptibility to Environmental Hazards convened by the Scientific Group on Methodologies for the Safety Evaluation of Chemicals (SGOMSEC) held DNA repair processes, although such traits have not yet been found in the human population. On the other hand, studies of individual differences in xenobiotic metabolism have revealed an increasing number of polymorphic enzymes that are involved in the metabolism of carcinogens [bib_ref] Genetic polymorphisms of cytochromes P450 as a biomarker of susceptibility to environmental..., Hong [/bib_ref].
Biotransformation, involving metabolic activation and detoxification, plays a central role in determining the ultimate effects of exposure to chemical carcinogens. Depending on the enzyme, there may be one or several mutant alleles that produce enzyme variants with reduced or increased efficiency in comparison with the wild-type form. In some cases, such as the null genotypes of glutathione S-transferases M 1 (GSTMI) and Ti (GSTTI), the functional enzyme is completely missing [bib_ref] The role of human glutathione transferases and epoxide hydrolases in the metabolism..., Seidegard [/bib_ref].
Numerous epidemiological studies have indicated an over-representation of some metabolic genotypes or phenotypes in various forms of cancer, suggesting that those individuals are excessively prone to such diseases [bib_ref] Genetic polymorphisms of cytochromes P450 as a biomarker of susceptibility to environmental..., Hong [/bib_ref]. With the possible exception of tobacco smoking in, for example, lung cancer associated with the GSTMI and cytochrome P450 (CYP) polymorphisms, epidemiology is not usually able to exactly pinpoint the exposures that are responsible for the findings, as the critical influence of carcinogen-metabolizing enzymes occurs several years before exposure-associated cancer develops.
The question of individual sensitivity to genotoxic carcinogens can also be viewed by studying the involvement of genetic polymorphisms in determining genotoxic response after exposure to specific carcinogens. For this purpose, methods based on genotoxicity assays with human cells, such as cytogenetic techniques, are very useful as they can be applied to any exposures known to produce such alterations. Thus, no specific method development for each new exposing agent is required, and the net effect of complex exposures can be evaluated. Differences between genotypes can be assessed by comparing cytogenetic responses after in vitro exposure or in individuals exposed to the genotoxins of interest in vivo.
The present review will concentrate on the application of cytogenetic biomarkers-chromosomal aberrations (CAs), sister chromatid exchanges (SCEs), micronuclei (MN), and numerical chromosome changes-in studies of individual susceptibility related to polymorphisms of carcinogen-metabolizing enzymes.
## Cytogenetic parameters
The four cytogenetic parameters already mentioned-CAs, SCEs, MN, and numerical chromosome alterations-are all based on microscopic analyses of cell specimens, but each of them measures a different genotoxic phenomenon. In humans, cytogenetic alterations are most often scored from peripheral lymphocytes after they have been stimulated to divide in culture by a mitogen, e.g., phytohemagglutinin [bib_ref] Human peripheraf blood lymphocytes for the analysis of chromosome aberrations in mutagen..., Evans [/bib_ref].
CAs are structural alterations, breaks and rearrangements, in chromosomes, usually observed in metaphase-blocked cells using conventional microscopy [bib_ref] Human peripheraf blood lymphocytes for the analysis of chromosome aberrations in mutagen..., Evans [/bib_ref]. In principle, chromosomal aberrations can be divided into chromosome-type lesionsproduced by ionizing radiations and some chemical clastogens in Go-GI-stage cellsand chromatid-type lesions that ionizing radiation causes in S-G2-stage cells and that are the main type of aberrations induced by most (S-phase dependent) chemical clastogens. Chromosome-type rearrangements, such as translocations and dicentrics inspected in biological dosimetry of radiation, can also be analyzed using chromosome painting based on fluorescence in situ hybridization (FISH) with chromosome-specific DNA probe libraries [bib_ref] Rapid human chromosome aberration analysis using fluorescence in situ hybridization, Lucas [/bib_ref]. Recently, a simplified FISH method to score chromosome breakage and alterations of chromosome number, using tandem DNA probes specific for a region in chromosome number 1, was reported (7). Polymerase chain reaction-based methods for the analysis of lymphocyte-specific illegitimate chromosome recombination involving human immunoglobulin or immune receptor loci and considered to depict genetic instability have also been described [bib_ref] Occurrence of bcl-2 oncofene translocation with increased frequency in the peripheral b..., Bell [/bib_ref]. In two recent independent reports, increased rates of CAs in peripheral lymphocytes were shown to be associated with later development of cancer [bib_ref] Cancer risk in humans predicted by increased levels of chromosomal aberrations in..., Hagmar [/bib_ref] [bib_ref] Are chromosome aberrations in circulating lymphocytes predictive of future cancer onset in..., Bonassi [/bib_ref]. Thus, the analysis of CAs is presently considered to be the cytogenetic method of choice in studies of human exposure to genotoxic carcinogens. A wide range of genotoxins have shown CA induction in vitro and in vivo. Known in vivo inducers of CAs in humans indude ionizing radiations, alkylating cytostatics, tobacco smoking, benzene, and styrene.
SCEs represent symmetrical exchanges of DNA segments between the sister chromatids of a duplicated metaphase chromosome and (because the sister chromatids are supposed to be equal) are not themselves mutations. SCEs are formed during the S-phase of the cell cycle and are efficiently induced by, for example, UV light and many chemical genotoxins, especially the S-phase dependent clastogens [bib_ref] Sister-chromatid exchange: second report of the Gene-Tox program, Tucker [/bib_ref]. The fact that each cell examined yields an SCE score makes the technique a very powerful tool for the assessment of differences between genotypes, particularly in vitro. In human lymphocytes in vivo, tobacco smoking, alkylating cytostatics, and ethylene oxide are well-documented SCE inducers.
Micronuclei are small additional nuclei observable in interphase cells. They are formed from acentric chromosomeor chromatid-type fragments and whole chromosomes that have lagged behind in cell division, being left outside both daughter nuclei [bib_ref] Micronuclei as an index of cytogenetic damage: past, present, and future, Heddle [/bib_ref]. Because of their dual origin (fragments or whole chromosomes), MN induction can be triggered by either clastogens or agents influencing the mitotic apparatus, such as spindle poisons. As cell division is a prerequisite for MN formation, MN in cultured peripheral lymphocytes are usually analyzed from cytokinesis-blocked cells, to identify cells that have divided once in culture [bib_ref] Measurement of micronuclei in lymphocytes, Fenech [/bib_ref]. MN can also be scored in exfoliated cells of buccal, nasal, esophageal, bronchial, or urothelial mucosa [bib_ref] The use of the micronucleus test on exfoliated cells to identify anticlastogenic..., Rosin [/bib_ref]. The presence of whole chromosomes in MN can be checked by identifying centromeric DNA sequences or kinetochore proteins in the MN [bib_ref] The identification of micronucleated chromosomes: a possible assay for aneuploidy, Thomson [/bib_ref] [bib_ref] Detection of whole chromosomes in micronuclei of cytokinesis-blocked human lymphocytes by antikinetochore..., Norppa [/bib_ref]. MN analysis appears to be a good in vitro tool to investigate the effects of clastogens and agents (aneuploidogens) that induce numerical abnormalities of chromosomes. In vivo, increased MN frequencies have been associated with exposure to ionizing radiation, aging, and gender [bib_ref] Micronucleus assay in lymphocytes as a tool to biomonitor human exposure to..., Norppa [/bib_ref]. In buccal or nasal mucosa, MN induction has clearly been shown for various ethnic tobacco-chewing habits [bib_ref] The use of the micronucleus test on exfoliated cells to identify anticlastogenic..., Rosin [/bib_ref] and exposure to formaldehyde [bib_ref] Micronucleated cells in nasal mucosa of formaldehyde-exposed workers, Ballarin [/bib_ref].
The development of FISH techniques has made it possible to detect, in a very simple manner, the copy numbers of any specific human chromosome for which a centromeric DNA probe is available [bib_ref] Advantages and limitations of using fluorescence in situ hybridization for the detection..., Eastmond [/bib_ref]. Both aneuploid and polyploid cells are identified, although hyperdiploids cannot usually be distinguished from polyploids unless probes for more than two chromosomes are used. The tandem FISH assay mentioned above provides information on both structural and numerical chromosomal aberrations [bib_ref] Detection of chromosomal breakage in the lcen-1q12 region of interphase human lymphocytes..., Rupa [/bib_ref]. Such analyses have thus far been used as biomarkers in humans only in a few cases, although they can be applied, besides peripheral lymphocytes, to any human tissue available.
## Genetic polymorphisms and cytogenetic assays in vrtro
The involvement of a polymorphism in determining the cytogenetic effects of a specific genotoxin can easily be tested in vitro in cultured cells collected from donors representing different genotypes or phenotypes. A requirement is, of course, that the cells used adequately express the polymorphic condition, i.e., in the case of metabolic genes, the enzyme in question.
In the examples available, peripheral lymphocytes have been used, which are also the most convenient choice, because of easy availability and well-established culture techniques [fig_ref] Table 1: Results of in vitro studies on the influence of genetic polymorphisms of... [/fig_ref].
The first example of the utilization of the in vitro approach was the study of Wiencke et al. [bib_ref] Human glutathione S-transferase deficiency as a marker of susceptibility to epoxide-induced cytogenetic..., Wiencke [/bib_ref] in which the effects of two isomeric epoxides, trans-stilbene oxide and cis-stilbene oxide, on SCEs were studied in cultured lymphocytes from persons able or unable to conjugate trans-stilbene oxide with glutathione. This trait reflects the genetic polymorphism of GSTM1 that is expressed in the leukocytes of the conjugators (corresponding to the GSTMI-positive genotype, with at least one copy of the intact gene) but is totally absent from the nonconjugators (the GSTMI-null genotype having a homozygous deletion of the gene). The nonconjugators were clearly more sensitive than the conjugators to SCE induction by trans-stilbene oxide, a substrate of GSTM 1; no clear difference was observed between the phenotypes in tests with cis-stilbene oxide, which is not a good substrate for GSTM1-mediated glutathione conjugation also appeared to be involved in the case of 1,2-epoxy-3-butene [bib_ref] Influence of GSTMJ genotype on sister chromatid exchange induction by styrene-7,8-oxide and..., Uuskiila [/bib_ref] , as lymphocytes of GSTMI-null donors were more sensitive than GSTMI-positive donors to SCE induction by this epoxide metabolite of 1,3-butadiene. Another epoxide, styrene 7,8-oxide, a metabolite of styrene, showed the same SCE induction in both genotypes, and appeared, therefore, not to be dependent on the GSTM1-catalyzed detoxification [bib_ref] Influence of GSTMJ genotype on sister chromatid exchange induction by styrene-7,8-oxide and..., Uuskiila [/bib_ref]. The GSTM1 genotype (or phenotype) did not influence SCE induction by two other reactive metabolites of 1,3-butadiene-1,2:3,4-diepoxybutane and 3,4-epoxybutene-1,2-diol (23-25).
Hallier et al. [bib_ref] Polymorphism of glutathione conjugation of methyl bromide, ethylene oxide and dichloromethane in..., Hallier [/bib_ref] described another polymorphism, identified by the presence or absence of methyl bromide glutathione conjugation in erythrocytes. After treatment of whole-blood samples, the nonconjugators' lymphocytes showed SCE induction by methyl bromide, methylene chloride, and ethylene oxide while no such effects could be seen in the conjugators. The phenotypes identified probably represented GSTT1-positive (at least one copy of the gene) and GSTTI-null (gene homozygously deleted) genotypes, as GSTT1 is an erythrocytic enzyme.
The GSTT1 polymorphism was recently also shown to explain individual differences in sensitivity to 1,2:3,4-diepoxybutane (DEB), a diepoxide metabolite of 1,3-butadiene. Early studies of Wiencke et al. [bib_ref] Individual susceptibility to induced chromosome damage and its implications for detecting genotoxic..., Wiencke [/bib_ref] demonstrated that humans can be dassified as DEB sensitive (about 20% of the persons studied) or DEB resistant on the basis of SCE induction by DEB in cultured lymphocytes. The DEB-sensitive donors also showed an enhanced induction of CAs by DEB [bib_ref] Bimodal distribution of sensitivity to SCE induction by diepoxybutane in human lymphocytes...., Wiencke [/bib_ref] and had an elevated baseline rate of SCEs [bib_ref] Bimodal distribution of sensitivity to SCE induction by diepoxybutane in human lymphocytes...., Kelsey [/bib_ref]. Recent expansions of these studies have demonstrated that all [bib_ref] Role of GSTTI and GSTMI genotypes in determining individual sensitivity to sister..., Norppa [/bib_ref] [bib_ref] Sister chromatid exchanges, glutathione S-transferase e deletion and cytogenetic sensitivity to diepoxybutane..., Kelsey [/bib_ref] [bib_ref] Repeated analysis of sister chromatid exchange induction by diepoxybutane in cultured human..., Landi [/bib_ref] or the majority (67%) (32) of the DEB-sensitive donors have the GSTTJnull genotype. Lymphocytes from GSTTInull donors also showed an enhanced induction of MN by DEB in vitro [bib_ref] Role of glutathione S-transferase TI and MI genotypes in determining individual sensitivity..., Norppa [/bib_ref]. In our experiments, erythrocytes from DEBsensitive donors had no detectable GSTT1 activity [bib_ref] Role of GSTTI and GSTMI genotypes in determining individual sensitivity to sister..., Norppa [/bib_ref] , and the SCE responses of GSTTI-null and positive individuals showed no overlapping [bib_ref] Role of GSTTI and GSTMI genotypes in determining individual sensitivity to sister..., Norppa [/bib_ref] [bib_ref] Repeated analysis of sister chromatid exchange induction by diepoxybutane in cultured human..., Landi [/bib_ref] [bib_ref] Influence of erythrocyte glutathione S-transferase TI on sister chromatid exchanges induced by..., Pelin [/bib_ref].
It is presently unclear what the DEB-sensitive GSTTI-positive individuals described by Wiencke et al. [bib_ref] Gene deletion of glutathione S-transferase: correlation with induced genetic damage and potential..., Wiencke [/bib_ref] actually represent. A point mutation resulting in a loss of GSTT1 activity, which would not be detected by usual GSTTI genotyping, has been described by Warholm et al. [bib_ref] Genotypic and phenotypic determination of polymorphic glutathione transferase Ti in a Swedish..., Warholm [/bib_ref] ; this mutation was, however, very rare (2 among 270 donors) in the Swedish population studied. In cultures of purified lymphocytes-with no erythrocytes and no GSTT1-the DEB-resistant (GSTTI-positive) donors showed very similar SCE induction as the DEB-sensitive (GSTTInull) donors [bib_ref] Influence of erythrocyte glutathione S-transferase TI on sister chromatid exchanges induced by..., Pelin [/bib_ref] , and the addition of erythrocytes from a resistant person to the purified lymphocytes of a sensitive person removed the DEB sensitivity [bib_ref] Effect of red cells and plasma blood in determining individual lymphocytes sensitivity..., Landi [/bib_ref].
In Salmonella typhimurium transfected with human GSTTI, the expression of GSTT1 activity increased the mutagenicity of DEB, thus functioning as a metabolic activation route [bib_ref] Human glutathione S-transferase TI-1 enhances mutagenicity of 1,2-dibromoethane, dibromomethane and 1,2,3,4-diepoxybutane in..., Thier [/bib_ref]. Therefore, it would appear that glutathione conjugation of DEB leads to enhanced genotoxicity in (bacterial) cells, where the target DNA is present, while the same reaction in erythrocytes of whole-blood lymphocyte cultures prevents DEB from reacting with DNA. Although epidemiologic studies have shown several examples of increased disease risk in GSTTl-null but not in GSTTI-positive individuals (3) it may also be possible that GSTTI activates some tissueor cell-specific mutagens in humans [bib_ref] Human glutathione S-transferase theta (GSTTI): cDNA cloning and the characterization of a..., Pemble [/bib_ref].
Our recent findings suggested that the GSTT1 nulls also show higher SCE induction by an in vitro treatment with styrene 7,8-oxide, although the effect was not nearly as dramatic as with DEB [bib_ref] Polymorphisms of xenobiotic metabolizing enzymes: influence on cytogenetic parameters in vitro and..., Norppa [/bib_ref]. This result appears to agree with the fact that glutathione conjugation is a minor pathway in styrene 7,8-oxide detoxification. However, the proposed main detoxification enzyme of styrene 7,8-oxide, microsomal epoxide hydrolase, is also polymorphic [bib_ref] Human microsomal epoxide hydrolase: genetic polymorphism and functional expression in vitro of..., Hassett [/bib_ref] , albeit the significance of this polymorphism in determining epoxide hydrolase levels in humans is still unclear. It must be kept in mind in all studies correlating biological effects with genetic polymorphisms that differences in mean biological responses between genotypes are not necessarily attributable to that particular genotype, but may also be due to such uncontrolled factors as other unknown or unchecked polymorphisms, acquired differences in enzyme levels, or experimental variation.
The deficiency of low Km aldehyde dehydrogenase (ALDH2), resulting in impaired detoxification of acetaldehyde, is common in Oriental populations. About one-half of all Japanese are either heterozygotes (ALDH21/ALDH22) or homozygotes (ALDH22IALDH22) for a mutation in the ALDH2 gene [bib_ref] Low 4 aldehyde dehydrogenase (ALDH2) polymorphism, alcohol-drinking behavior, and chromosome alterations in..., Morimoto [/bib_ref]. Takeshita [bib_ref] Low 4 aldehyde dehydrogenase (ALDH2) polymorphism, alcohol-drinking behavior, and chromosome alterations in..., Morimoto [/bib_ref] observed that lymphocytes of ALDH2-deficient donors are more sensitive than those of ALDH2-proficient individuals (ALDH2I/ALDH2i) to SCE induction by an in vitro treatment with hydroquinone, a metabolite of benzene. The genotype effect was higher in habitual daily alcohol drinkers than in those who consumed alcohol less frequently. The authors did not provide an explanation for these findings.
Another aspect, aside from the use of the in vitro cytogenetic assays to identify dependence on genetic polymorphisms, is the influence of genotype data on routine genotoxicity testing using human cells. For instance, lymphocytes from GSTM1null donors will respond higher to certain treatments than individuals with the GSTMJ-positive genotype, and differential responses obtained, e.g., between human whole-blood and isolated lymphocyte cultures, may partly be explained by the GSTT1 activity of red blood cells not present in purified lymphocyte cultures. In critical cases, such as those shown by Hallier et al. [bib_ref] Polymorphism of glutathione conjugation of methyl bromide, ethylene oxide and dichloromethane in..., Hallier [/bib_ref] for methyl bromide, methylene chloride, and ethylene oxide, one phenotype (conjugators) will give a negative result and another (nonconjugators) positive.
## Individual susceptibility and cytogenetic markers in vivo
The few studies available on genetic polymorphism and the rate of cytogenetic alterations in humans in vivo have almost exclusively been performed using peripheral lymphocytes. Thus far, the studies have addressed induced or baseline cytogenetic damage in association with polymorphisms of GSTMI, GSTTI, cytochrome P450 1AI (CYPIA1), cytochrome P4502D6 (CYP2D6), and N-acetyltransferase 2 (NA T2).
The first investigation describing an association between genetic polymorphisms and cytogenetic response to a genotoxic exposure in man in vivo was the report of van Poppel et al. [bib_ref] Increased cytogenetic damage in smokers deficient in glutathione S-transferase isozyme p, Van Poppel [/bib_ref] on a small increase in SCEs in heavy smokers not expressing GSTM1, as compared with GSTMl-proficient heavy smokers; the effect was not observed in moderate smokers or nonsmokers. MN in sputum cells were not influenced by smoking or the GSTMI phenotype (43). Cheng et al. [bib_ref] Glutathione S-transferase p genotype, diet, and smoking as determinants of sister chromatid..., Cheng [/bib_ref] [bib_ref] Comparison of sister chromatid exchange in peripheral lymphocytes in lung cancer cases..., Cheng [/bib_ref] [bib_ref] Increased micronucleus frequency in lymphocytes from smokers with lung cancer, Cheng [/bib_ref] observed a significant association between the GSTMI-null genotype and elevated SCE frequencies (but not MN) in lymphocytes of healthy control persons (but not of lung cancer patients). However, the finding appeared not to be associated with smoking. CYPIAI MspI and Ile-Val mutations and CYP2D6G to A mutations did not influence SCE frequencies among the controls [bib_ref] Glutathione S-transferase p genotype, diet, and smoking as determinants of sister chromatid..., Cheng [/bib_ref].
The influence of the GSTMI genotype on smoking-induced chromosome damage was further supported by our recent studies, which show a statistically significant association between the GSTMI-null genotype and elevated CA rates (but not SCE or MN) in lymphocytes of smoking Italian greenhouse workers and controls [bib_ref] Cytogenetic monitoring of occupational exposure to pesticides: characterization of GSTMI, GSTTI and..., Scarpato [/bib_ref] [bib_ref] Influence of GSTMI and GSTTI polymorphisms on the frequency of chromosome aberrations..., Scarpato [/bib_ref]. The effect particularly concerned chromatid-type aberrations. The pesticide exposure of the greenhouse workers had no significant influence on CAs, SCEs, or MN. No dependence of the cytogenetic biomarkers on GSTTI or NAT2 genotypes was observed, except for a slightly higher baseline SCE level among GSTT1-positive donors in comparison with GSTTI-null individuals; this finding was possibly due to chance, as there were only four GSTTI-null subjects [bib_ref] Cytogenetic monitoring of occupational exposure to pesticides: characterization of GSTMI, GSTTI and..., Scarpato [/bib_ref].
The preliminary results of another recent study among Danish bus drivers indicated an increasing gradient of CA frequencies from GSTMI-positivelNAT2 fast acetylator genotype to GSTMI-null/ NA T2 slow acetylator genotype [bib_ref] Polymorphisms of xenobiotic metabolizing enzymes: influence on cytogenetic parameters in vitro and..., Norppa [/bib_ref]. This finding suggested that these genotypes modulate the genotoxic effects of exposures (diesel exhaust) experienced by the bus drivers in their work.
Carstensen et al. [bib_ref] B-and T-lymphocyte micronuclei in chimney sweeps with respect to genetic polymorphism for..., Carstensen [/bib_ref] and Ichiba et al. [bib_ref] Aromatic DNA adducts, micronuclei and genetic polymorphism for CYPIAJ and GSTI in..., Ichiba [/bib_ref] studied MN in T-and B-lymphocytes of chimney sweeps, but could not observe any significant association between MN frequency and GSTMI and CYPIAI genotypes, in connection with the occupational exposure or smoking. Although the GSTMI genotype itself was not significantly associated with variations in MN frequency, correlation between the frequency of MN in T-lymphocytes and aromatic DNA adducts in total white blood cells was statistically significant among GSTMI-null individuals (sweeps and controls combined) [bib_ref] Aromatic DNA adducts, micronuclei and genetic polymorphism for CYPIAJ and GSTI in..., Ichiba [/bib_ref]. CYPIA1 MspI and Ile-Val polymorphisms did not influence MN frequencies, although the common "noninducible" MspI genotype mllml showed significantly higher DNA adduct levels than the tentative high-inducible ml/m2 genotype [bib_ref] Aromatic DNA adducts, micronuclei and genetic polymorphism for CYPIAJ and GSTI in..., Ichiba [/bib_ref].
There are also studies suggesting no influence of GSTMI genotype on cytogenetic damage in human lymphocytes, but these data derive from control cultures established for in vitro studies not designed to reveal differences in baseline rate of cytogenetic damage [bib_ref] Human glutathione S-transferase deficiency as a marker of susceptibility to epoxide-induced cytogenetic..., Wiencke [/bib_ref] [bib_ref] Influence of GSTMJ genotype on sister chromatid exchange induction by styrene-7,8-oxide and..., Uuskiila [/bib_ref] [bib_ref] Role of GSTTI and GSTMI genotypes in determining individual sensitivity to sister..., Norppa [/bib_ref] [bib_ref] Bimodal distribution of sensitivity to SCE induction by diepoxybutane in human lymphocytes...., Wiencke [/bib_ref].
The clear difference in sensitivity to DEB (metabolite of 1,3-butadiene) in vitro in association with the GSTTI genotype (above) has also triggered in vivo cytogenetic studies on a possible genotype effect among 1,3-butadiene production workers. The preliminary results of a European study showed an increase in CA in lymphocytes of GSTTI-null workers in comparison with GSTTI-positive workers [bib_ref] Assessment of exposure to butadiene in the process industry, Sorsa [/bib_ref]. A study in the United States on SCEs in 1,3-butadiene producers, however, failed to show any association with the GSTTI genotype [bib_ref] Sister chromatid exchanges, glutathione S-transferase e deletion and cytogenetic sensitivity to diepoxybutane..., Kelsey [/bib_ref]. Due to automated processes, exposure to butadiene in the facilities studied was fairly low. The positive European study suggested that genotype determination may improve the sensitivity of cytogenetic assays in detecting the effects of low-level genotoxic exposures.
The American study did confirm the higher background SCE frequency of GSTTI-null subjects as compared with GSTTI-positive individuals [bib_ref] Sister chromatid exchanges, glutathione S-transferase e deletion and cytogenetic sensitivity to diepoxybutane..., Kelsey [/bib_ref]. Wiencke et al. [bib_ref] Gene deletion of glutathione S-transferase: correlation with induced genetic damage and potential..., Wiencke [/bib_ref] calculated that the DEB sensitivity and the GSTTI genotype were the most important factors influencing baseline SCE frequency, explaining 37 and 27%, respectively, of the variation observed. Smoking, for instance, was found to explain only 6 to 16% of the variation. The genotype effect was independent of smoking and was thought to reflect exposure to internal or ubiquitous external factors dependent on glutathione conjugation [bib_ref] Gene deletion of glutathione S-transferase: correlation with induced genetic damage and potential..., Wiencke [/bib_ref]. One candidate could be ethylene oxide, a tentative substrate of GSTT1 [bib_ref] Polymorphism of glutathione conjugation of methyl bromide, ethylene oxide and dichloromethane in..., Hallier [/bib_ref] , formed by xenobiotic metabolism from endogenous ethylene. CA count was not significantly elevated in the lymphocytes of the DEB-sensitive subjects [bib_ref] Bimodal distribution of sensitivity to SCE induction by diepoxybutane in human lymphocytes...., Wiencke [/bib_ref]. and Takeshita (41) observed that ALDH2-deficient habitual alcohol drinkers had higher mean frequency of SCEs in lymphocytes than ALDH2-proficient individuals who consumed alcohol daily. This effect probably reflected the higher acetaldehyde levels found in the peripheral blood of the ALDH2-deficient subjects who also showed an elevated level of acetaldehyde adducts in hemoglobin [bib_ref] Low 14, aldehyde dehydrogenase (ALDH2) polymorphism and chromosome alterations in alcohol drinkers, Morimoto [/bib_ref]. Other lifestyle factors may also play a role, since a significant genotype effect could be shown in smokers but not in nonsmokers [bib_ref] Low 4 aldehyde dehydrogenase (ALDH2) polymorphism, alcohol-drinking behavior, and chromosome alterations in..., Morimoto [/bib_ref]
# Conclusions
The relationship between human genetic polymorphisms and genotoxicity is a new field of research and only a limited amount of information is presently available. Already, the data available suggest that donor genotype can have dramatic influence on induced or baseline levels of cytogenetic alterations both in vitro and in vivo.
The use of in vitro assays to test the impact of specific genotypes or phenotypes on the cytogenetic effects of genotoxins is highly recommended as it will give an idea about the biological significance of genotypic differences, which is not revealed, for example, by enzyme activity measurements alone. In this way, in vitro experiments can serve as a guideline for selecting exposures and genotypes to be studied in vivo. A prerequisite is, of course, that the polymorphisms of interest are expressed in the cells. Adequate expression has been shown at least for GSTM1 in human lymphocytes and GSTT1 in erythrocytes. The relationship between the GSTTJ genotype and sensitivity to SCE induction by DEB seems to be a good example of an exceptionally clear association.
Understanding the significance of genetic polymorphisms in determining genotoxic response will also have an important influence on requirements concerning the use of human cells in genotoxicity testing. Some discrepancies between negative and positive findings or dramatically different active concentration ranges will be explained by different genotypes of the donors.
Studies of cytogenetic biomarkers among exposed humans show that the determination of polymorphisms is becoming an increasingly important aspect that may make the assays more sensitive and more specific in identifying the effect and the sensitive subgroups. At present, it seems that at least GSTM1 and GSTTI genotypes of the donors should be studied on a routine basis when in vivo cytogenetic effects are concerned. Rarer genotypes can be evaluated only in large populations-even the 10 to 20% prevalence of the GSTTI-null genotype among Caucasians may be too low to allow evaluation if the exposure groups are small. Besides good knowledge in genetic toxicology and molecular genetics, expertise in epidemiology is becoming quite useful in planning such exercises, and statistical treatment will not be valid without multivariate analyses in which all of the variables can be duly taken into account.
There will also be much to be learned in risk assessment. Restricted still in most cases to surrogate tissues, such as peripheral lymphocytes, instead of the real targets of carcinogenesis, many things can go wrong in assessing the risk. For instance, the expression or role of the critical metabolizing enzymes may vary in different tissues. From these considerations, one arrives at the ethical issues that are dealt with more thoroughly elsewhere in this issue [bib_ref] Ethical, social, and legal issues surrounding studies of susceptible populations and individuals, Soskolne [/bib_ref]. It is obvious that the cytogeneticist is faced with ethical questions both in sample collection and when the results of the research are used in risk estimation. It can be gathered from what has been presented above that sound conclusions about the role of genetic polymorphisms in determining genotoxic risks can be achieved only when the complex issue has been studied in depth.
Information obtained from cytogenetic studies of genetic polymorphisms can be used to recognize the genotoxically relevant substrates of the polymorphic enzymes, to identify susceptible genotypes, and to improve the sensitivity of cytogenetic assays both in vitro and in vivo. Such knowledge can be utilized in mechanistic studies and cancer epidemiology. In search for the role of genetic polymorphisms in carcinogenesis, the cytogenetic markers form a link in the chain of evidence which stretches from experimental in vitro work, through analyses of exposed humans, to studies on cancer. Genetic polymorphisms can be followed in all of these steps.
[table] Table 1: Results of in vitro studies on the influence of genetic polymorphisms of carcinogen-metabolizing enzymes on the induction of chromosomal aberrations, sister chromatid exchanges, and micronuclei in cultured human lymphocytes. ALDH2, aldehyde dehydrogenase 2; CAs, chromosomal aberrations; DEB, 1,2:3,4-diepoxybutane; DEB sensitivity, enhanced SCE response to 1,2:3,4-diepoxybutane; EBD, 3,4-epoxy-1,2-butanediol; GSTM1, glutathione S-transferase Mi; GSTT1, glutathione S-transferase Ti; MN, micronuclei; SCEs, sister chromatid exchanges. "Genotype or phenotype giving an enhanced cytogenetic response. [/table]
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Past climate variations recorded in needle-like aragonites correlate with organic carbon burial efficiency as revealed by lake sediments in Croatia
www.nature.com/scientificreports/ indicating cooler conditions. The Sr/Ca ratio of aragonite is typically used as a proxy for sea surface temperature (SST) variability 16 because incorporation of Sr into aragonite coral skeletons as well as in inorganic aragonite is temperature dependent, i.e., an increase in temperature lowers the Sr/Ca ratio in inorganic aragonite 17,18 and coral skeletons 19 . However, this simplistic interpretation has been challenged because it has been proven that incorporation of Sr into aragonite corral skeletons is also affected by vital effects 20,21 , variability of the Sr/Ca ratio in the oceans 22,23 and unwanted effects caused by algal symbionts 24 . Compared with numerous paleoclimate studies of biogenic aragonite, the paleoclimate potential of inorganic aragonite in marine/lake sediments needs to be explored, which is mainly because sedimentary environments in which inorganic aragonite precipitates are rare, and studies show that the Sr/Ca ratio of inorganic aragonite is much less sensitive to temperature changes than the Sr/Ca ratio of coral aragonite skeletons 21 . Despite this obstacle, the main advantage of inorganic aragonite is the lack of vital effects. We validated the reliability of this novel proxy by also acquiring the same kind of data for Core M-2 that was taken from a different basin of the same lake. An age-depth model of Core M1-A(Fig. 2)was previously described 25 . The significance of the Sr/Ca ratio as a paleotemperature proxy was also confirmed by correlation with other previously published studies, including the main Holocene climatic events from the wider Mediterranean region 26-31 .Results and discussionStudy site and core description. Lake Veliko jezero, with a surface area of 1.44 km 2 , consists of three basins; core M1-A is derived from the deepest basin, with a maximum depth of 46 m(Fig. 1). Core M2 was retrieved from the second largest basin from a depth of 40 m. The lake is located on the island of Mljet (42°44´ N; 17°31´E), which is a part of the Adriatic carbonate platform 32 (AdcP). Late Jurassic and Early Cretaceous dolomites are exposed around the lake. The lake is part of the karst depression system, which is now a submerged sinkhole due to Holocene sea level rise. At the early Holocene, 10.8 cal ka BP 25 , because of the sea level rise and the presence of permeable karst, a wetland formed that transitioned into a brackish lake and finally ended as a marine lake because of sea flooding through the Soline channel (2.5 m deep) 2.3 cal ka BP 25 . The lake was perennial, with an almost constant salinity of ̴ 25‰ during the studied interval 33 (8.3-2.6 cal ka BP). The only connection with the sea was through permeable karst, while the only source of freshwater was local precipitation.Core M1-A recovered from Lake Veliko jezero is 417 cm long, measured from the lake bottom, and four lithological units are distinguished(Fig. 2). The top unit (0-108 cm) is made up of gray homogenous marine sediment. The underlying unit (108-241 cm) consists of deep lake sediment characterized by alternations of
The drivers of organic carbon (OC) burial efficiency are still poorly understood despite their key role in reliable projections of future climate trends. Here, we provide insights on this issue by presenting a paleoclimate time series of sediments, including the OC contents, from Lake Veliko jezero, Croatia. The Sr/Ca ratios of the bulk sediment are mainly derived from the strontium (Sr) and calcium (Ca) concentrations of needle-like aragonite in Core M1-A and used as paleotemperature and paleohydrology indicators. Four major and six minor cold and dry events were detected in the interval from 8.3 to 2.6 calibrated kilo anno before present (cal ka BP). The combined assessment of Sr/Ca ratios, OC content, carbon/nitrogen (C/N) ratios, stable carbon isotope (δ 13 C) ratios, and modeled geochemical proxies for paleoredox conditions and aeolian input revealed that cold and dry climate states promoted anoxic conditions in the lake, thereby enhancing organic matter preservation and increasing the OC burial efficiency. Our study shows that the projected future increase in temperature might play an important role in the OC burial efficiency of meromictic lakes.
Lakes have disproportionally large annual amounts of buried organic carbon (OC) compared to oceans 1 and are of great importance for the global carbon budget and cycle; thus, they might also have a vast impact on climate change in the future. However, the influence of climate on OC burial efficiency is still not precisely understood. The influence of temperature on the OC burial efficiency has been studied extensively, mainly for lakes at higher northern latitudes [bib_ref] Temperature-controlled organic carbon mineralization in lake sediments, Gudasz [/bib_ref] [bib_ref] Climate change predicted to cause severe increase of organic carbon in lakes, Larsen [/bib_ref] [bib_ref] Land-use change, not climate, controls organic carbon burial in lakes, Anderson [/bib_ref] [bib_ref] Low organic carbon burial efficiency in arctic lake sediments, Sobek [/bib_ref] [bib_ref] Large increases in carbon burial in northern lakes during the Anthropocene, Heathcote [/bib_ref]. Although a large number of studies have focused on this topic, a consensus has not been reached on the possible influence of temperature on the OC burial efficiency. Moreover, the main driving mechanisms that are directly responsible for the observed changes in OC content of lake sediments have not been identified. Studies have inferred that higher temperatures enhance OC burial mainly as a consequence of denser vegetation cover 3,7-10 , with an increase in temperature positively correlated with OC mineralization [bib_ref] Temperature-controlled organic carbon mineralization in lake sediments, Gudasz [/bib_ref] [bib_ref] Organic carbon burial efficiency in lake sediments controlled by oxygen exposure time..., Sobek [/bib_ref] [bib_ref] Temperature sensitivity of organic carbon mineralization in contrasting lake sediments, Gudasz [/bib_ref]. In more recent studies, anthropogenic influences have been reported, primarily through the role of reactive nitrogen and phosphorus on OC burial efficiency [bib_ref] Land-use change, not climate, controls organic carbon burial in lakes, Anderson [/bib_ref] [bib_ref] Large increases in carbon burial in northern lakes during the Anthropocene, Heathcote [/bib_ref] [bib_ref] Anthropogenic alteration of nutrient supply increases the global freshwater carbon sink, Anderson [/bib_ref] , and the temperature effect has been negated. Furthermore, oxygen exposure time and redox conditions in the water may also play a prominent role in the OC burial efficiency [bib_ref] Low organic carbon burial efficiency in arctic lake sediments, Sobek [/bib_ref] [bib_ref] Organic carbon burial efficiency in lake sediments controlled by oxygen exposure time..., Sobek [/bib_ref] [bib_ref] Oxygen dynamics control the burial of organic carbon in a eutrophic reservoir, Carey [/bib_ref] [bib_ref] Hot tops, cold bottoms: Synergistic climate warming and shielding effects increase carbon..., Bartosiewicz [/bib_ref].
The majority of previous studies on OC burial efficiency have focused on recent and subrecent lake sediments, where climate effects can easily be concealed by anthropogenic influences. Additionally, in latitudinal studies, the temperature effect can be masked by other variables, such as changes in vegetation cover and/or precipitation rate. To infer the climate influence on OC burial efficiency, we studied sediments of Lake Veliko jezero, Croatia , for the period 8.3 to 2.6 cal ka BP. In detail, we interpreted the OC content in the context of high-resolution relative paleoclimate (log ratio of Sr/Ca) and paleoredox proxies (log ratio of Mo/detrital elements) and as an indicator for past aeolian activity (log ratio of Zr over Al). Our approach for correlating paleoclimate and paleoredox proxies was performed by considering the properties of compositional data, which enabled more precise interpretations. The Sr/Ca ratios of the bulk sediment are mainly derived from Sr and Ca concentrations of needle-like aragonite in Core M1-A and used as paleoclimate, temperature proxies, with higher ratios www.nature.com/scientificreports/ white and dark laminas mainly composed of aragonite and organic matter. The top part of that unit consists of a few centimeters of oxidized lake sediment. The following unit from 241 to 343 cm is marsh to shallow lake sediment. Finally, the bottom unit (343-417 cm) is terra rossa-type soil interbedded with thick tephra. Core M2 resembles Core M1-A with the difference that unit boundaries are at different depths. Therefore, the studied interval spans from 127 to 266 cm in this core.
## M1-
High-resolution XRF scanning and age-depth model. The Sr/Ca records were obtained by highresolution XRF core scanning at split core surfaces at 1 cm and 2 mm resolutions. We compared the Sr/Ca data and ratios between the two investigated cores (M1-A and M2) to confirm the robustness of our record and exclude any potential analytical artifacts. M1-A core chronology is based on four tephra layers and four C-14 dates [bib_ref] Holocene tephra record of Lake Veliko jezero, Croatia : implications for the..., Razum [/bib_ref]. Briefly, the studied interval chronology is very well constrained since in ̴ 1.25 m, there are three tephra layers, which serve as depth-age model anchor points: two charcoal C-14 dates free of reservoir effects and one marine shell C-14 date with known reservoir effects [fig_ref] Figure 2: Age-depth model for Core M1-A from Lake Veliko jezero, with core images... [/fig_ref]. The geochronological age of Core M2 is based on one plant remaining sample, which was dated by radiocarbon methodology. Three additional data points were included in the age-depth model of this core. Two of these ages are based on visible tephra layers correlated to the known volcanic eruptions previously recognized in Core M1-A [bib_ref] Holocene tephra record of Lake Veliko jezero, Croatia : implications for the..., Razum [/bib_ref]. The third date corresponds to a time of marine intrusion into the lake, which is marked by a sharp boundary between the top and underlying lithological units [fig_ref] Figure 2: Age-depth model for Core M1-A from Lake Veliko jezero, with core images... [/fig_ref] in both cores. It was radiometrically dated in Core M1-A 25 . Individual radiocarbon dates were calibrated using the R package rbacon(the age-depth model for core M2 is presented in the Supplementary file, [fig_ref] Figure 4: Sr/Ca [/fig_ref]. Despite the lower resolution of the Core M2 record, it is evident that major peaks and troughs in Sr/Ca are well presented in both datasets [fig_ref] Figure 3: Sr/Ca records for Cores M1 and M2 as indicators of relative paleotemperature... [/fig_ref].
Sr/Ca ratio as paleoclimate proxy. Our working hypothesis was that the Sr/Ca ratio in bulk sediment almost exclusively reflects the Sr/Ca ratio of needle-like aragonite in the sediment [fig_ref] Figure 2: Age-depth model for Core M1-A from Lake Veliko jezero, with core images... [/fig_ref]. The temperature and Mg/Ca ratio are the two most important variables for the precipitation of inorganic aragonite [bib_ref] Influences of temperature and Mg: Ca ratio on CaCO3 precipitates from seawater, Morse [/bib_ref]. The main source of Mg for the aragonites from the Veliko jezero lake could be seawater, and the second source could be from the surrounding carbonate rocks, which are mainly dolomites. Although the Mg/Ca ratio does not affect the crystallization of aragonites directly, it prevents the formation of calcite and the transformation of aragonite into calcite [bib_ref] The role of magnesium in the crystal growth of calcite and aragonite..., Berner [/bib_ref] [bib_ref] Element partitioning during precipitation of aragonite from seawater: A framework for understanding..., Gaetani [/bib_ref]. Additionally, the Sr/Ca ratio in aragonite is not affected by variations in salinity, sulfate or carbon dioxide content [bib_ref] The role of magnesium in the crystal growth of calcite and aragonite..., Berner [/bib_ref] [bib_ref] The solubility of calcite and aragonite in sulfate-free seawater and the seeded..., Mucci [/bib_ref] [bib_ref] Calcite and aragonite precipitation from seawater solutions of various salinities: precipitation rates..., Zhong [/bib_ref]. Needle-like aragonite currently precipitates during late spring and summer in the adjacent Lake Malo jezero [bib_ref] Whiting events and the formation of aragonite in Mediterranean karstic marine lakes:..., Sondi [/bib_ref]. Since the Sr/Ca ratio of needle-like aragonite is largely temperature dependent 17,18 , we assume that the Sr/Ca ratio of the studied sediment could be used as a relative paleotemperature proxy. Mineralogical analysis performed with X-ray diffraction (XRD) and scanning electron microscopy coupled with www.nature.com/scientificreports/ energy dispersive spectroscopy (SEM-EDS) proved that inorganic needle-like aragonite is the main mineral and nearly the only carbonate phase in our investigated samples [fig_ref] Figure 2: Age-depth model for Core M1-A from Lake Veliko jezero, with core images... [/fig_ref]. The only exception is an interval from 201 to 214 cm, where inorganic rhombohedral Mg-calcite is the main carbonate phase, thus confirming previously published results [bib_ref] Holocene lake development of two dalmatian lagoons (Malo and Veliko Jezero, Isle..., Wunsam [/bib_ref]. According to the age model of Core M1-A, the occurrence of Mg-calcite coincides with known time intervals of wet climate during which lake levels were high and generally correspond to pluvial periods observed in the wider Mediterranean region (Figs. 3 and 4, from ca. 7.6 to 7 ka BP) [bib_ref] Holocene vegetation and climate changes in the central Mediterranean inferred from a..., Combourieu-Nebout [/bib_ref] [bib_ref] Paleohydrology reconstruction and Holocene climate variability in the South Adriatic Sea, Siani [/bib_ref] [bib_ref] Lateglacial to Holocene trace element record (Ba, Mg, Sr) from Corchia Cave, Regattieri [/bib_ref] [bib_ref] Enhanced rainfall in the Mediterranean region during the last Sapropel Event, Kallel [/bib_ref] [bib_ref] Mid-holocene climate variations revealed by high-resolution speleothem records from soreq cave, israel..., Bar-Matthews [/bib_ref] [bib_ref] Contrasting patterns of precipitation seasonality during the Holocene in the south-and north-central..., Magny [/bib_ref] [bib_ref] Stratigraphic evidence for a ' pluvial phase' between ca 8200-7100 ka from..., Zhornyak [/bib_ref]. We propose that during this period, increased freshwater input lowered the Mg/Ca ratio in the lake water, leading to Mgcalcite precipitation and hindering the precipitation of aragonite. This consequently led to a Sr/Ca decrease in our records because Mg-calcite incorporates Sr in the crystal lattice less effectively than aragonite [bib_ref] Measurements of distribution coefficients of strontium and barium between carbonate precipitate and..., Kitano [/bib_ref] ; thus, the Sr/ Ca ratio of bulk sediment cannot be interpreted as a relative paleotemperature proxy in this interval. However, the predominance of Mg-calcite over aragonite points to wetter climate conditions, which are also observed in the wider region during this time period [bib_ref] Holocene vegetation and climate changes in the central Mediterranean inferred from a..., Combourieu-Nebout [/bib_ref] [bib_ref] Paleohydrology reconstruction and Holocene climate variability in the South Adriatic Sea, Siani [/bib_ref] [bib_ref] Lateglacial to Holocene trace element record (Ba, Mg, Sr) from Corchia Cave, Regattieri [/bib_ref] [bib_ref] Enhanced rainfall in the Mediterranean region during the last Sapropel Event, Kallel [/bib_ref] [bib_ref] Mid-holocene climate variations revealed by high-resolution speleothem records from soreq cave, israel..., Bar-Matthews [/bib_ref] [bib_ref] Contrasting patterns of precipitation seasonality during the Holocene in the south-and north-central..., Magny [/bib_ref] [bib_ref] Stratigraphic evidence for a ' pluvial phase' between ca 8200-7100 ka from..., Zhornyak [/bib_ref].
To utilize the Sr/Ca ratio as a relative paleotemperature proxy, we have proven that detrital Sr and Ca inputs are negligible. XRD analyses revealed aragonite as the main mineral, with minor quartz content limited only to the oldest portion of the studied period. Furthermore, large variances of the centered log ratio transformed variables of Sr and Ca relative to detrital elements, such as titanium (Ti) and aluminum (Al) (Supplementary file, [fig_ref] Figure 2: Age-depth model for Core M1-A from Lake Veliko jezero, with core images... [/fig_ref] combined with small logratio varaince of Sr and Ca relative to the inorganic carbon (INC) (Supplementary file, [fig_ref] Figure 2: Age-depth model for Core M1-A from Lake Veliko jezero, with core images... [/fig_ref] additionally confirmed that Sr and Ca represent carbonate components and are not of detrital origin.
Finally, the Sr budget of the Lake Veliko jezero water and consequently the Sr/Ca ratio of the bulk sediment can also be influenced by hydrological variability. Changes in hydrological regime would theoretically affect the relative marine influence at this location because of a limited connection of the lake to the Adriatic Sea through permeable karst: ocean water is characterized by higher Sr concentrations than freshwater [bib_ref] Elemental mass-balance of material carried by major world rivers, Martin [/bib_ref]. Two possible scenarios emerge if hydrologically induced Sr availability was the limiting factor for the Sr/Ca ratio of the bulk sediment. MD 90-917 [bib_ref] Holocene vegetation and climate changes in the central Mediterranean inferred from a..., Combourieu-Nebout [/bib_ref] www.nature.com/scientificreports/ First, during cold periods, Sr/Ca in the lake water would be lower because of the reduced evaporation rate, i.e., decreased marine influence, which would finally result in a relative decrease in the Sr concentration of the lake water and consequently the Sr/Ca of the lake sediments. The opposite would be the case for warmer climate conditions. This scenario, however, can be discarded based upon the good correlation of the maxima in our Sr/ Ca record with cold events that were previously observed in multiple paleoclimate archives in the Mediterranean region [fig_ref] Figure 3: Sr/Ca records for Cores M1 and M2 as indicators of relative paleotemperature... [/fig_ref]. In [fig_ref] Figure 3: Sr/Ca records for Cores M1 and M2 as indicators of relative paleotemperature... [/fig_ref] , the locally estimated scatterplot smoothed (LOESS) Sr/Ca curve, with a smoothing factor of 0.09, displays four distinct peaks centered at 8.3, 6.0, 4.25 and 2.9 cal ka BP. The first Sr/Ca maxima in our record, which are centered at 8.3 ka, are coeval with cold events described in the pollen record from the Adriatic Sea 28 and a decrease in the sea surface temperature 29 recognized in the same core. In the Alboran Sea, a drop in the temperature during that time period was recorded as well [bib_ref] Variability of the western Mediterranean Sea surface temperature during the last 25000..., Cacho [/bib_ref] [bib_ref] Mid-Holocene emergence of a lowfrequency millennial oscillation in western Mediterranean climate: Implications..., Fletcher [/bib_ref] , while a minor drop in the temperature was also observed in Gemini Lake 31 . These events correlate with the North Atlantic cold event (NAC5) [bib_ref] Persistent solar influence on north atlantic climate during the Holocene, Bond [/bib_ref]. Following the pluvial period (7.6 to 7 cal ka BP) characterized by Mg-calcite deposition instead of aragonite, another maximum in the Sr/Ca record from the M1-A core, which is centered at approximately 6 cal ka BP, can be correlated with cold spells recognized in the Adriatic 27,28 and Tyrrhenian and less clearly in the Alboran Sea 26 as well as the NAC4 event in the North Atlantic 51 . Temperature reconstructions based on chironomid communities from Gemini and Verdarolo lakes also indicate cold conditions at approximately 6 cal ka BP 31 . A Sr/Ca maximum at approximately 4.25 cal ka BP is correlated with a decrease in the temperature in the Adriatic Sea and Italian lakes [bib_ref] Holocene vegetation and climate changes in the central Mediterranean inferred from a..., Combourieu-Nebout [/bib_ref] [bib_ref] Warm Mediterranean mid-Holocene summers inferred from fossil midge assemblages, Samartin [/bib_ref] and in the north [bib_ref] Persistent solar influence on north atlantic climate during the Holocene, Bond [/bib_ref]. Finally, an increase in the Sr/Ca centered at approximately 2.9 cal ka BP correlates well with the cold spells already recognized in the Adriatic, Alboran and Tyrrhenian seas [bib_ref] Variability of the western Mediterranean Sea surface temperature during the last 25000..., Cacho [/bib_ref] [bib_ref] Holocene seasonal sea-surface temperature variations in the southern Adriatic Sea inferred from..., Sangiorgi [/bib_ref] [bib_ref] Holocene vegetation and climate changes in the central Mediterranean inferred from a..., Combourieu-Nebout [/bib_ref] [bib_ref] Mid-Holocene emergence of a lowfrequency millennial oscillation in western Mediterranean climate: Implications..., Fletcher [/bib_ref] as well as in the North Atlantic 51 . A less pronounced temperature decrease during this time interval was observed in Lake Gemini, Italy [bib_ref] Warm Mediterranean mid-Holocene summers inferred from fossil midge assemblages, Samartin [/bib_ref].
The second scenario implies that the environment during cold events was also dry, while during warmer events, it was wetter, which would lead to a decreased relative marine influence and the dilution of the Sr budget in the lake water. Such changes would finally result in lower Sr/Ca during warm periods and higher values during cold periods. To further investigate the role of wet versus dry conditions as potential drivers of Sr/Ca variability, we studied additional chemical elements acquired by XRF scanning, such as Br, Rb, Si, K, Na, Al, Fe, Mn, Zr, Ti and Mo, which were primarily validated by wet-chemical analysis on discrete samples, e.g., through inductively coupled plasma mass spectrometry (ICP-MS). We used all of these elements to model proxies called balances; for paleotemperature, aeolian input and paleoredox conditions via orthonormal log ratio transformation (OLR) [bib_ref] Compositional data: the sample space and its structure, Egozcue [/bib_ref] , which enabled firm statistical parameters to be established for reliable interpretations of the involved processes 53 . Sr/Ca (detrended) July air temperature(°C) C/N www.nature.com/scientificreports/ The methodology is fully explained in a previous study [bib_ref] A compositional approach to the reconstruction of geochemical processes involved in the..., Razum [/bib_ref] and described briefly in the Methods section. Ultimately, five balances were modeled, each of which is a proxy for a certain process: b3 and b4 are proxies for paleoredox conditions, b5 is a proxy for aeolian activity, and b2 is a proxy for paleoclimate Sr/Ca (rationale for the balance construction is shown in the Supplementary file, text S1, and sign . The data analyses show that an increase in Sr/Ca correlates with increases in anoxic conditions (r (b2-b3) = 0.55, p (0.05) = 0.00001) and aeolian activity (r (b2-b5) = 0.56, p (0.05) = 0.00001) (Supplementary file, table S2) [fig_ref] Figure 4: Sr/Ca [/fig_ref]. A comparison between b2 and Sr/Ca is presented in the Supplementary file, [fig_ref] Figure 3: Sr/Ca records for Cores M1 and M2 as indicators of relative paleotemperature... [/fig_ref]. Furthermore, a speleothembased paleohydrological reconstruction from Corchia cave in Italy 30 implies that during the time corresponding to the Sr/Ca maxima in our record, climate conditions were generally drier [fig_ref] Figure 4: Sr/Ca [/fig_ref]. However, it is also evident that the Sr/Ca ratios in our record do not exhibit exceptionally high values during the most widespread dry event in the Mediterranean and wider region at 4.2 ka [bib_ref] Holocene seasonal sea-surface temperature variations in the southern Adriatic Sea inferred from..., Sangiorgi [/bib_ref] [bib_ref] Mid-holocene climate variations revealed by high-resolution speleothem records from soreq cave, israel..., Bar-Matthews [/bib_ref] [bib_ref] A pronounced dry event recorded around 4.2 ka in brine sediments from..., Arz [/bib_ref] [bib_ref] Late Holocene drought responsible for the collapse of Old World civilizations is..., Drysdale [/bib_ref] [bib_ref] Possible complexity of the climatic event around 4300-3800 cal, Magny [/bib_ref] [bib_ref] The 42 ka BP Event in the Mediterranean region: An overview, Bini [/bib_ref]. This finding implies that hydrological conditions probably played less of a role in driving Sr/Ca ratios than the temperature effect. Following all lines of evidence, we propose that the Sr/Ca ratio of Lake Veliko jezero bulk sediment represents the Sr/Ca ratio of inorganic needle-like aragonite, which mainly reflects relative paleotemperature changes, while hydrological variability likely plays a secondary role.
One of the main advantages of XRF core scanning in addition to being nondestructive is the high resolution. If Sr/Ca ratios are a temperature indicator, then they may also be applicable for short events; for example, we observe several relatively brief cold events at 8.0, 6.6, 5.4, 4.8, 4.0 and 3.3 cal ka BP. From these, the events at 8.0, 6.6, 5.4 and 4.0 cal ka BP were also recorded in Lake Gemini while the event at 3.3 ka BP was recognized in Lake Verdarolo [bib_ref] Warm Mediterranean mid-Holocene summers inferred from fossil midge assemblages, Samartin [/bib_ref]. Most of these events are characterized by an increase in anoxic conditions and aeolian input, indicating not only cooler but also drier climate conditions at our sites [fig_ref] Figure 4: Sr/Ca [/fig_ref]. These events can be detected due to the combined effects of the limited lake size and detrital influence on the studied lake(s) and the high resolution of the data. The small size of this lake (surface area of 1.44 km 2 ) has very limited detrital influence, i.e., small effects of internal and landscape filters 58 result in increased sensitivity in recording smaller-scale climate events.
## Disentangling the drivers of oc burial efficiency.
To decipher the potential drivers of OC burial efficiency, we analyzed the OC, inorganic carbon (INC) and organic nitrogen (N) and explored their relationship with enhanced anoxic episodes, cold spells and aeolian input. Additionally, to better characterize the provenance of organic matter (OM), we analyzed δ 13 C throughout the same interval.
Higher C/N ratios (> 10) indicate potential mixing of land-derived and autochthonous organic matter 59,60 , while more negative δ 13 C values could also be related to the increase in the land-derived component of organic matter [bib_ref] A review of coastal palaeoclimate and relative sea-level reconstructions using δ13C and..., Lamb [/bib_ref]. Our results demonstrate a slight long-term decreasing trend in the C/N record that coincides with a much stronger increasing trend of δ 13 C values. This anticorrelated pattern of the two proxies indicates that land-derived organic matter is partly decreasing, which is consistent with the overall detrital influence during our studied interval [fig_ref] Figure 4: Sr/Ca [/fig_ref]. Additional evidence comes from the occurrence of quartz, which is only found at the base of the studied interval. A combination of two factors is most likely responsible for the described trends. First, lake deepening caused by the Holocene sea level rise moved the shoreline away from the core site. More specifically, the distance between Core M1-A and the Pomena doline, which is a small terra rossa soil patch adjacent to Lake Veliko jezero (Pomena field, , increased. Second, due to the Holocene sea level rise, a gradual increase in marine influence through permeable karst occurred. Sea level rise changed the lake biota [bib_ref] Holocene lake development of two dalmatian lagoons (Malo and Veliko Jezero, Isle..., Wunsam [/bib_ref] and consequently the organic carbon content and composition.
During the studied interval, only two different pollen zones occurred, one with Juniperus and Phillyrea ca. 8 to 6.5 ka BP and another with Quercus ilex from 6.5 ka BP to the present [bib_ref] New palynological and tephrostratigraphical investigations of two salt lagoons on the island..., Jahns [/bib_ref]. This finding implies minor changes in terrestrial vegetation with negligible impacts on carbon content, composition and variability.
The correlation of the OC content with the Sr/Ca record reflecting temperature variations on the millennial time scale suggests that the OC content increased during cold exposure [fig_ref] Figure 4: Sr/Ca [/fig_ref]. The observed OC increase during cold events is in line with a previous study 2 , where a temperature decrease leads to low mineralization of OC. However, the temperature effect through OC mineralization was not a substantial factor for OC preservation in the Lake Veliko jezero sediments. If temperature is a driver of OC preservation on millennial time scales, then cold climate conditions would decrease the degradation rate of algal (autochthonous) organic matter [bib_ref] C / N ratios in a sediment core from Nakaumi Lagoon, southwest..., Sampei [/bib_ref] , which would result in lower C/N values, higher OC and more positive δ 13 C values, which we did not observe in our data. Indeed, we observe slightly higher C/N and more negative δ 13 C values during cold spells, which would imply that land-sourced organic matter increased during cold spells as a result of enhanced aeolian input as confirmed by correlation analyses r(b2-b5) [fig_ref] Figure 4: Sr/Ca [/fig_ref]. However, we argue that an increase in land-sourced organic matter is not the main mechanism underlying the overall OC increase because a more than double increase in OC during cold spells would have caused a substantial increase in C/N, which is not observed in our record. An exception is the 4.2 event, when maximum C/N ratios occurred.
The OC amount and variability also correlate with the paleoredox proxy [fig_ref] Figure 4: Sr/Ca [/fig_ref] , i.e., an increase in anoxic conditions corresponds to an increase in OC content. Based on this finding, we propose that an increase in anoxic conditions is the main factor that led to OC preservation at our site. A decrease in temperature and possibly drier conditions during cold events would shift the redox zone boundary and thermocline closer to the surface 64 , which would prevent mixing of the water throughout the water body, thereby causing the anoxic boundary to move upward and leaving the majority of the water column under anoxic conditions. This supposition is also confirmed by the high correlation between paleotemperature (b2) and paleoredox (b3) proxies. Although the depth of a thermocline depends on a number of factors, such as the lake size, dissolved organic content, temperature, wind activity, etc. [bib_ref] Effects of thermocline deepening on lake plankton communities, Cantin [/bib_ref]. We believe that the temperature was the key factor controlling the thermocline and redox zone boundary depth in Lake Veliko jezero. These findings are underpinned by a study of modern processes in Lake www.nature.com/scientificreports/ Veliko jezero lake, where the thermocline occurs only during summer months [bib_ref] Ecological characteristics of the Mljet Island seawater lakes ( South Adriatic Sea..., Benović [/bib_ref]. Finally, a shallow thermocline/ redox zone boundary would cause most of the organic matter produced in and transported into the lake to be prevented from decomposing, resulting in higher OC values during cold spells.
# Conclusions
The data presented here demonstrate that temperature changes may have a significant impact on OC burial efficiency. The temperature decrease and likely drier climate conditions caused shifts in the anoxic boundary towards the surface of the lake and thus prevented OC mineralization in an oxic environment. Compared with many previous studies, this study is unbiased with respect to anthropogenic influences, latitude changes or significant vegetation changes, which might have an effect on OC burial efficiency. Our results demonstrate that climate variability was able to trigger mechanisms inherent to the lake, thereby resulting in oscillations in OC burial efficiency. The Sr/Ca ratio of bulk sediment reflects the formation of aragonite needles in this special lake setting and is a novel approach that can be utilized for paleoclimate reconstructions. We were able to identify several cooling events observed in the wider Mediterranean area, although the unique high resolution of our data enabled us to also identify a number of short-term cold and dry events throughout the 8.3 to 2.6 cal ka BP period that have mainly not been previously observed. Further high-resolution studies on additional archives would be beneficial for investigating their wider regional character.
# Methods
Collection and extended description of sediment cores. Sediment cores were recovered from the deepest part of the Veliko jezero (M1-A at -45 m) and the deepest part of the second largest basin in Veliko jezero (M2 at -40 m) using a 3 m long piston corer (60 mm diameter UWITEC piston corer) deployed from a floating platform. The M1-A core had a total length of 459 cm, while the M2 core had a total length of 406 cm. Before being split lengthwise and photographed, the entire cores were subjected to magnetic susceptibility loop sensor measurements at 2 cm intervals with a Bartington MS2 magnetic susceptibility system. The split cores were logged for their lithology (smear slides), grain size, textures, structures, clast size and color using both the Munsell color chart and diffuse reflectance measurements (CIELAB-International Commission on Illumination L*a*b*) at 1 cm intervals using an X-rite DTP22 digital swatchbook spectrophotometer. Additionally, the magnetic susceptibility values of the split cores were determined using a Bartington MS2E system at 1 cm intervals. Working halves were subsampled at 1 cm intervals and stored at + 4 °C in plastic bags until further analysis, while the archived halves were stored in D-tubes in a cold chamber at the same temperature. For this study, we used intervals from 114 to 240 cm in core M1-A and from 127 to 266 cm in core M2.
High-resolution XRF scanning. Both cores M1-A and M2 were analyzed at XRF Core Scanner III (AVAATECH Serial No. 12) at the MARUM Center for Marine Environmental Sciences at the University of Bremen, Germany. Core M1-A was scanned at resolutions of 1 and 0.2 cm, while for Core M2, data were collected every 1 cm. In both cases, generator settings of 10 and 30 kV were applied. A current of 1 mA was used at 30 kV, and 0.2 mA and 0.5 mA at 10 kV were used at 1 cm and 0.2 cm intervals, respectively. The sampling times were 20 s and 30 s for the 1 cm and 0.2 cm intervals, respectively. Before scanning, the split core surface was covered with a 4 µm thin SPEXCerti Prep Ultralene foil to avoid contamination of the XRF measurement unit as well as desiccation of the sediment. The data reported here were acquired by a Canberra X-PIPS Silicon Drift Detector (SDD; Model SXD 15C-150-500) with a 150 eV X-ray resolution, a Canberra Digital Spectrum Analyzer DAS 1000 and an Oxford Instruments 100 W Neptune X-ray tube with rhodium (Rh) target material. Raw data spectra were processed by the analyzing the X-ray spectra by the Iterative Least square software (WIN AXIL) package from Canberra Eurisys. Reliable data (expressed in counts per second (cps)) were collected for the following elements: Br, Rb, Sr, Zr, Al, Si, S, K, Ca, Ti, Mn, and Fe; however, Mo values were considered unreliable because the concentration was close to or below the instruments' detection limit, and they were validated with an additional ICP-MS analysis on selected samples (Supplementary file, . For each core, the RGB and LAB color parameters were obtained at 68 μm resolution. The analysis was performed within 24 h after the core was opened. Semiquantitative X-ray diffraction and scanning electron microscopy. To explore whether nonaragonite carbonate phases are present in the studied sediment samples, we performed XRD analyses on samples from Core M1-A. To cover the complete record from this core, we analyzed the mineral composition of 42 composite samples that were defined by homogenization of 3 cm long intervals. The analyses were performed on a PANalytical X'Pert Powder X-ray diffractometer equipped with Ni-filter CuKα radiation, a vertical goniometer with θ/θ geometry and a PIXcel detector. The scan conditions were as follows: 45 kV and 40 mA, ¼ divergence slit and anti-scatter slits, 0.02° 2θ step size, and 2 s time per step. After the samples were ground with mortar and pestle, they were sieved through a 0.4 mm sieve. To reduce the grain size of the material to < 5 μm, powdered samples were ground in McCrone micronizing mills. XRD digital scans were analyzed using the Philips X'Pert High Score search and match function to identify peaks and determine qualitative mineral compositions. Additionally, black and white laminas were examined throughout the cores with scanning electron microscopy (SEM) to study the morphology of aragonite minerals to test their inorganic origin. In the interval from 201 to 214 cm, SEM information was used to infer the morphology and potential nonbiogenic origin of Mg-calcite, which was proven by both XRD and SEM-EDS (energy-dispersive X-ray spectroscopy). and total nitrogen (TN) of the sediment samples were analyzed using a Thermo Scientific FLASH 2000 Series Nitrogen and Carbon analyzer of the Croatian Geological Survey at a 1 cm resolution in the studied interval. This method allows for direct measurements of the total carbon (TC) and TN. The addition of HCl removes the carbonate component and allows for the determination of TOC. Carbonate was removed by treating 1 g of sediment sample with 12 ml 4 M HCl and heating in a centrifuge tube sitting in a hot block for 2 h. The insoluble residue was washed with Milli-Q water and centrifuged (2x), freeze-dried and weighed. The carbon content of the insoluble residue after HCl treatment is the TOC. The difference between TC and TOC was used for the calculation of TIC, whereas the calcium carbonate (CaCO 3 ) content was calculated from the obtained TIC values. The C/N mass ratio was calculated by dividing the TOC and TN. XRD analyses of insoluble residue were used to confirm that carbonates were not present in the samples after HCl treatment. A split of the acid-washed sample was weighed into tin capsules optimized for stable carbon and nitrogen isotope ratio measurements in the Sample Weight Calculator of the Stable Isotope Facility (SIF) at the University of California, USA. The samples were shipped to SIF in sealed evaporation plates with 96 wells. The sediments at SIF were analyzed for δ 13 C and δ 15 N using an Elementar Vario EL Cube (Elementar Analysensysteme GmbH, Hanau, Germany) interfaced with an Isoprime VisION IRMS (Elementar UK Ltd, Cheadle, UK). The isotope data are expressed relative to international standards VPDB (Vienna Pee Dee Belemnite). The long-term standard deviation reported by the SIF is 0.2‰ for δ 13 C and δ 0.3‰ for δ 15 N. Stable carbon isotopes were measured at a 10 cm resolution.
Statistical methods. Geochemical data obtained with XRF are compositional data, i.e., all components are parts of the same whole [bib_ref] The Statistical Analysis of Compositional Data, Aitchison [/bib_ref]. It is very hard to measure all elements; therefore, in reality, we analyzed a subcomposition, i.e., only some of all possible parts. Prior to the statistical analyses, one should represent data that are originally given as elements of a simplex space in log ratio coordinates. Orthonormal log ratio coordinates (olr) were used [bib_ref] Compositional data: the sample space and its structure, Egozcue [/bib_ref] [bib_ref] Isometric logratio for compositional data analysis, Egozcue [/bib_ref] to construct geochemical proxies [bib_ref] A compositional approach to the reconstruction of geochemical processes involved in the..., Razum [/bib_ref]. The orthonormal basis for the olr compositional biplots was constructed using the CoDa pack [bib_ref] CoDaPack 2.0: a stand-alone, multi-platform compositional software, Comas [/bib_ref]. The construction of the sample basis was conducted by performing sequential binary partitioning (SBP) of a compositional vector. Prior to the proxy construction, the dimensionality of the problem was reduced with the aid of a compositional biplot [bib_ref] Biplots of compositional data, Aitchison [/bib_ref] , which helped in discarding redundant elements, i.e., those that carry geochemically similar information (have small variance between clr transformed variables). Proxies constructed via this method are free of compositional data restrictions regarding the multivariate statistics and correlation analyses. It is important to stress that the high Mg-calcite interval (group High D) was removed from the correlation analysis of constructed proxies reported in the main paper because of inherently different geochemical affiliations. The rationale for balance construction and compositional biplot interpretation is presented in the Supplementary information.
## Data availability
The datasets generated during and/or analyzed during the current study are available in the PANGAEA repository, https:// doi. org/ 10. 1594/ PANGA EA. 924331
[fig] Figure 2: Age-depth model for Core M1-A from Lake Veliko jezero, with core images and lithological units shown next to the depth scale. SEM images of inorganic aragonite and Mg-calcite are shown on the right-hand side.Scientific Reports | (2021) 11:7568 | https://doi.org/10.1038/s41598-021-87166-2 [/fig]
[fig] Figure 3: Sr/Ca records for Cores M1 and M2 as indicators of relative paleotemperature and paleohydrology compared to paleotemperature and paleohydrological proxies from other records from the wider Mediterranean region. The Mg-calcite-rich interval is underlain by a dark blue area in the Sr/Ca record. Otherwise, needle-like aragonite is the dominant phase. NAC, AdC, AC, and TC are abbreviations for cold events based on the original publications. [/fig]
[fig] Figure 4: Sr/Ca (3-point average curve) balances b3 (redox) and b5 (aeolian input) derived from the XRF data and its connection with organic matter data (C/N, OC and δ 13 C). Gray bands indicate the main cold events, and purple bands are minor cold events recognized in M1-A. The correlation of those events with temperature changes in Lakes Gemini and Verdarolo and the humidity index from Corchia are visible on the right side of the figure. Scientific Reports | (2021) 11:7568 | https://doi.org/10.1038/s41598-021-87166-2 [/fig]
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Case report: A novel truncating variant of BCL11B associated with rare feature of craniosynostosis and global developmental delay
# Introduction
Craniosynostosis is the premature fusion of one or more of the calvarial sutures. The incidence of craniosynostosis is estimated to be 1 in 2000-2500 live births. In the cases of syndromic craniosynostosis, a series of clinical phenotypes, such as skull expansion, midfacial hypoplasia, increased intracranial pressure and dysmorphologies, are observed [bib_ref] Bcl11b regulates sutural patency in the mouse craniofacial skeleton, Kyrylkova [/bib_ref]. The major etiology of craniosynostosis includes varieties of factors including genetic and environmental factors [bib_ref] Clinical genetics of craniosynostosis, Wilkie [/bib_ref]. Moreover, genetic factor has been recognized as a nonnegligible etiology of craniosynostosis. In a 13-year birth cohort study, one-quarter of patients with craniosynostosis could be identified as having a genetic cause [bib_ref] Clinical genetics of craniosynostosis, Wilkie [/bib_ref]. An early genetic diagnosis can provide valuable information for intervention. For the treatment of craniosynostosis, patients need long-term strategies, including surgery such as open cranial reconstruction, minimally invasive strip craniectomy with spring implantation, cranial distraction and a postoperative molding helmet.
The transcription factor BCL11B (B Cell Leukemia 11B), encoding Cys2-His2 zinc-finger protein transcription factor, is located on chromosome 14q32.2. BCL11B is broadly expressed and has critical functions in regulating multiple systems including the central nervous system, immune system, and skin [bib_ref] Bcl11b prevents fatal autoimmunity by promoting Treg cell program and constraining innate..., Drashansky [/bib_ref]. BCL11B contains three types of functional structures: six C2HH zinc-finger binding domains (ZnF1-6), one C2HC zinc-finger binding domain, and a NuRD interacting domain [bib_ref] Bcl11 transcription factors regulate cortical development and function, Simon [/bib_ref]. The first report of BCL11B as a potential disease gene was in an infant bearing a de novo BCL11B missense mutation with severe combined immunodeficiency, craniofacial and dermal abnormalities, and the absence of corpus callosum [bib_ref] Multisystem anomalies in severe combined immunodeficiency with mutant Bcl11b, Punwani [/bib_ref]. Currently, 20 patients have been reported to harbor BCL11B pathogenic variants, The major clinical features include severe combined immunodeficiency, neurological disorders, dermal defects, congenital diaphragmatic hernia, and craniosynostosis. Recently, Goos et al. reported a de novo missense mutation in a patient with unilateral coronal suture craniosynostosis [bib_ref] A de novo substitution in Bcl11b leads to loss of interaction with..., Goos [/bib_ref]. However, it is rare about the description of craniosynostosis in patients with BCL11B mutations.
Here, we report a patient with a novel de novo frameshift variant exhibiting the combinational phenotype of craniosynostosis and developmental delay. Meanwhile, we review the clinical and genetic features of craniosynostosis patients with BCL11B mutations, which further delineate the phenotype and genotype of such patients.
# Materials and methods
Whole exome sequencing and Sanger sequencing Genomic DNA was extracted from peripheral blood using a whole blood genomic DNA extraction kit (Qiagen, German) according to the manufacturer's protocol. DNA fragments were enriched for exome sequences using the IDT xGen Exome Research Panel v2 (Integrated DNA Technologies, Coralville, IA, USA). According to our previous reports, sequencing was performed on NovaSeq 6000 (Illumina, San Diego, USA) according to protocols [bib_ref] Diagnostic and clinical utility of next-generation sequencing in children born with multiple..., Wang [/bib_ref]. The data analysis followed the pipeline established by our team [bib_ref] Clinical and genetic spectrum of a large cohort of children with epilepsy..., Yang [/bib_ref]. PhenoPro, including phenotype-driven analysis and variant-driven analysis, was performed to prioritize the disease-causing genes of Mendelian diseases as described before [bib_ref] Phenopro: a novel toolkit for assisting in the diagnosis of mendelian disease, Li [/bib_ref]. The pathogenicity was evaluated by the variation frequency, phenotypic coincidence with patients and the type of variation according to ACMG criteria [bib_ref] Standards and guidelines for the interpretation of sequence variants: a joint consensus..., Richards [/bib_ref]. The variant of the BCL11B gene was confirmed by Sanger sequencing on an ABI 3,730 Genetic analyzer (Applied Biosystems, Foster City, CA, USA).
# In silico analysis
Nonsense-mediated mRNA decay (NMD) efficacy of the BCL11B frameshift was evaluated using online NMDective resource (14). The wildtype 3-D protein structure of BCL11B was analyzed by the neural network AlphaFold, a new online source to predict the protein domain (15). The mutated amino acid sequence was acquired on the Mutalyzer website. Then we used SWISS-MODEL to predict the truncated protein structure according to the protein template from AlphaFold. All the protein structures were visualized by PyMOL 2.3.0.
# Results
## Case presentation
The boy is the first child of healthy non-consanguineous parents. He visited our hospital at 12 months of age with global developmental delay and recurrent allergy. He was born after uneventful full-term pregnancy with birth weight of 3,650 g (0.60 SD), and birth length of 52.0 cm (1.49 SD). He was observed with plagiocephaly, his birth head circumference is 33.2 cm (−0.99 SD) (Supplementary [fig_ref] FIGURE 1: Clinical features and sequence analysis [/fig_ref]. He had midfacial hypoplasia, hypertelorism, arched eyebrows, a long philtrum and a small mouth [fig_ref] TABLE 1: Clinical features of six craniosynostosis patients with mutations in BCL11B [/fig_ref]. His motor development milestones were delayed: he could not sit steadily at 14 months, he could walk with help at 18 months, and he could not clap his hands until 24 months, he could only say "baba" and "mama". Brain MRI at the age of 13 months showed widening of the extracerebral interval, dysmyelination of the bilateral periventricular white matter, abnormal signals of the cornu occipitale and a reduced right-brain cerebral volume [fig_ref] FIGURE 1: Clinical features and sequence analysis [/fig_ref] and [fig_ref] TABLE 1: Clinical features of six craniosynostosis patients with mutations in BCL11B [/fig_ref].
Further examinations, including brain CT and blood immunophenotyping analysis, were undertaken at the age of 25 months. Skull radiographs and three-dimensional computed tomography (3D-CT) showed craniosynostosis of the frontal and right-coronal suture fusion, and skull asymmetry was observed, with the eye axis deviating to the right side [fig_ref] FIGURE 1: Clinical features and sequence analysis [/fig_ref].
His immunophenotyping analysis results are shown in [fig_ref] TABLE 1: Clinical features of six craniosynostosis patients with mutations in BCL11B [/fig_ref] and [fig_ref] FIGURE 2: The pathogenic variant of BCL11B and the in silico prediction results [/fig_ref]. Notably, the number and percentage of eosinophil cells were increased to 910 cells/µl (normal range: 60-300 cells/µl) and 13.7% (normal range: 0.5-5%), respectively. The value of IgE in this patient was markedly increased to 944.91 IU/ml (normal range < 100 IU/ml).
[formula] Strabismus − − − − − Not tested Other − − Nystagmus − − − [/formula]
## Immune system function
[formula] IgE ↑ − NR − − − Eosinophil ↑ − NR − − − Frequent infection − − + − − − [/formula]
# Genetic analysis
WES was performed for the patient, and a novel heterozygous variant (NM_138576.4: c.2346_2361del, p.Gly783Ala6fs * 24) of the BCL11B gene was detected. The variant was located on the exon4, just before the ZnF4 domain [fig_ref] FIGURE 2: The pathogenic variant of BCL11B and the in silico prediction results [/fig_ref] and was predicted to generate a truncated protein with a lack of ZnF4-6 [fig_ref] FIGURE 2: The pathogenic variant of BCL11B and the in silico prediction results [/fig_ref]. The BCL11B gene variant was highly conserved and partly located in the ZnF4 domain [fig_ref] FIGURE 2: The pathogenic variant of BCL11B and the in silico prediction results [/fig_ref].
This frameshift variant was curated according to the ACMG criteria base on the following evidence: (i) NMDetective-A (score: 0.12) and NMDetective-B (score: 0) predicted the variant escaping NMD; the pLI (probability of being loss-of-function intolerance) score of BCL11B was 0.99, the observed/expected (o/e) ratio was 0.09, which suggests that BCL11B is highly possible of loss-of-function, PVS1_Strong; (ii) Sanger sequencing of parental samples confirmed that the variant was de novo [fig_ref] FIGURE 1: Clinical features and sequence analysis [/fig_ref] , and the phenotype was consistent with the BCL11B gene, PM6; (iii) the variant had not been recorded in gnomAD, the 1,000 Genomes database or our in-house database (43,000 individuals), PM2. (iv) we had submitted this variant to ClinVar database (VCV001184138.4) as pathogenic, and the lab of Institute of Human Genetics, University Hospital Muenster also submitted to ClinVar, with the evidence of PVS1 + PS2 + PS4, this same variant identified from a different patient. Therefore, the class of this variant was curated as pathogenetic.
We also reviewed currently reported 20 patients in literature. 18 patients carried 16 variations of the BCL11B gene, including ten frameshifts, four missense mutations and two nonsense mutations. The other two had chromosomal rearrangements resulting in diminished BCL11B expression (data not shown) [fig_ref] FIGURE 2: The pathogenic variant of BCL11B and the in silico prediction results [/fig_ref].
# Discussion
In this study, we report a novel de novo frameshift pathogenic variant (Gly783Ala fs * 24) in the BCL11B gene associated with craniosynostosis and developmental delay. Using the newly reported AlphaFold algorithm, a computational method that predicts protein structures with atomic accuracy (15), we predicted that this truncated protein would lack the last three C2HH zinc-finger domains (ZnF4-6). As BCL11B is a transcriptional activator, the premature stop-codon sequence of BCL11B may affect the protein's function in binding to its target DNA and its interactions with target proteins [bib_ref] Bcl11b: a new piece to the complex puzzle of amyotrophic lateral sclerosis..., Lennon [/bib_ref]. We curated the variant as pathogenic based on the ACMG criteria (PVS1_Strong + PS2 + PM6 + PM2). Although we predicted this frameshift variant escape NMD, whether the pathogenic mechanism of the variant is haploinsufficiency or gain of function remains unclear, and further experiment would be needed to address this. In the literature (17), the Cys81Leufs * 76 variant was predicted to activate NMD, while the Gly820Alafs * 27 variant was predicted to result in a protein with loss of DNA binding to the last C-terminus. Therefore, further studies are needed to elucidate the pathogenic mechanism of BCL11B gene.
In addition, this frameshift maybe a pathogenic hotpot of BCL11B gene as a second patient who carried the same frameshift mutation was recently submitted in the ClinVar database. Like our patient, this patient presented global developmental delay and the dysmorphic feature like trigonocephaly. However, no more detail clinical information was recorded in this patient. In our patient, we noticed that the number and percentage of eosinophils and the IgE values of our patient significantly increased. Interestingly, five cases with a high level of eosinophils were also reported by Lessel et al and Lu et al. [bib_ref] Bcl11b mutations in patients affected by a neurodevelopmental disorder with reduced type..., Lessel [/bib_ref] [bib_ref] A novel germline heterozygous Bcl11b variant causing severe atopic disease and immune..., Lu [/bib_ref]. Lu and associates proposed this germline BCL11Brelated atopic disease as a novel primary atopic disorder (PAD). The immunophenotyping of the patient revealed intact T-cell numbers as our patient presented (Supplementary [fig_ref] TABLE 1: Clinical features of six craniosynostosis patients with mutations in BCL11B [/fig_ref]. However, zebrafish experiment demonstrated that the BCL11B variant (p.Cys826Tyr) was unable to rescue T-cell development. Therefore, it would be important to explore how immune deficiency arose despite intact thymic development.
In a summary of the neurodevelopment disorder phenotype of all reported patients [bib_ref] A de novo substitution in Bcl11b leads to loss of interaction with..., Goos [/bib_ref] [bib_ref] Bcl11b mutations in patients affected by a neurodevelopmental disorder with reduced type..., Lessel [/bib_ref] [bib_ref] A novel germline heterozygous Bcl11b variant causing severe atopic disease and immune..., Lu [/bib_ref] [bib_ref] A De novo heterozygous frameshift mutation identified in Bcl11b causes neurodevelopmental disorder..., Homma [/bib_ref] [bib_ref] Mutant Bcl11b in a patient with a neurodevelopmental disorder and T-cell abnormalities, Yang [/bib_ref] , four were reported to present with abnormal MRI, exhibiting moderate ectopia of the amygdala [bib_ref] Bcl11b mutations in patients affected by a neurodevelopmental disorder with reduced type..., Lessel [/bib_ref] , hypoplasia of the globus pallidus [bib_ref] Bcl11b mutations in patients affected by a neurodevelopmental disorder with reduced type..., Lessel [/bib_ref] , callosal agenesis [bib_ref] Multisystem anomalies in severe combined immunodeficiency with mutant Bcl11b, Punwani [/bib_ref] , or an abnormal myelination pattern of white matter [bib_ref] Mutant Bcl11b in a patient with a neurodevelopmental disorder and T-cell abnormalities, Yang [/bib_ref]. Our patient had brain morphological abnormalities with brain asymmetry and affected growth of the right brain.
In this study, we reviewed the clinical phenotype of the reported patients with craniosynostosis and BCL11B variants (9, 24) and our patient (Table1). One de novo missense variant (c.7C > A, p. Arg3Ser) with craniosynostosis in BCL11B gene has been reported [bib_ref] A de novo substitution in Bcl11b leads to loss of interaction with..., Goos [/bib_ref]. The patient presented with sever craniosynostosis and received three corrective surgeries before the age of 13 years. Fortunately, the boy was otherwise healthy. Recently, another literature has reported four BCL11B missense mutations (p.Gly667Glu, p.Gly582Ser, p.Pro673Arg, p.Pro422Leu) with craniosynostosis or combined congenital diaphragmatic hernia (CDH) and craniosynostosis. All the BCL11B variants were inherited from their normal parents. The variant of c.1744G > A (p.Gly582Ser) was recorded in three individuals without craniosynostosis in our internal database and in one individual in gnomAD database. In this literature, clinical exome sequencing was performed in one patient and targeted sequencing of single gene (BCL11B) in other three patients, these sequence methods may miss real pathogenic gene variants. Therefore, the pathogenicity of these inherited missense BCL11B variants in craniosynostosis is questionable as the paper's title mentioned.
# Conclusion
In this study, our data revealed disruptions of BCL11B as a monogenic cause of craniosynostosis combined with a neurodevelopmental disorder. Our findings expand the genetic and phenotypic spectrum of the BCL11B gene in pediatric patients.
# Data availability statement
The datasets presented in this study can be found in online repositories. The names of the repository and accession number(s) can be found here: the GSA repository, accession number: subHRA004434.
# Ethics statement
The studies involving human participants were reviewed and approved by the ethics committees of the Children's Hospital, Fudan University (2015-130). Written informed consent to participate in this study and for the publication of any potentially identifiable images or data included in this article was provided by the participants' legal guardian/next of kin.
# Author contributions
XZ and BW contributed to the conception and design, data acquisition and analysis, and drafting of the manuscript. HC and YW performed the in silico analysis. PZ and YQ performed the data analysis of next-generation sequencing. GL performed the genetic experiment and sanger sequence analysis. XP collected clinical information and recorded the data. XD confirmed the clinical diagnosis and silico analysis. HW and CD contributed to the conception, design of this study, and revised the final manuscript. All authors contributed to the article and approved the submitted version.
# Funding
This work was supported by grants from the National Natural Science Foundation of China (grant no. 81471483).
[fig] FIGURE 1: Clinical features and sequence analysis. (A) Brain MR images. Abnormal signals of the cornu occipitale. (a) T1WI hypodensities and the smaller right brain volume; (b) T2WI hyperintensity and the widening of the extracerebral space; (c) increased FLAIR signals; (d) right-skewed midfacial hypoplasia. (B) Pedigree of the family and Sanger sequencing analysis. (C) CT images with 3D reconstruction. (a-b) Frontal seam and right coronal seam, (c) right apex seam, (d) left apex seam. The abnormal seam fusions are indicated by the red arrow, and the normal seam is indicated by the black arrow. [/fig]
[fig] FIGURE 2: The pathogenic variant of BCL11B and the in silico prediction results. (A) The localization of variants reported in the literature and the variant in our patient. The variant in bold red font is that of the patient in this report. (B) Structures of BCL11B WT analyzed by the AlphaFold network and BCL11B G783Afs * 24 predicted by SWISS-MODEL. The ZnF1-ZnF6 domains are marked in the corresponding colors of the schematic representations. (C) Sequence alignment across species with amino acid residues was performed, and our patient's mutated sequence is shown in bold red font. [/fig]
[table] TABLE 1: Clinical features of six craniosynostosis patients with mutations in BCL11B. [/table]
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Multimodal prehabilitation for major surgery in elderly patients to lower complications: protocol of a randomised, prospective, multicentre, multidisciplinary trial (PREHABIL Trial)
# Introduction background
The global volume of surgery has grown by about a third, from 234 million procedures in 2008 to more than 312 million in 2012, with most surgeries being performed in developed countries. Mortality rates are estimated to be between 1% and 4% in Europe. [bib_ref] Mortality after surgery in Europe: a 7 day cohort study, Pearse [/bib_ref] A minority of patients at high risk seem responsible for the majority of complications and mortality. [bib_ref] Identification and characterisation of the high-risk surgical population in the United Kingdom, Pearse [/bib_ref] It is, therefore, of key importance to identify patients at risk preoperatively for several reasons: it allows informed decisionmaking, it offers the opportunity for preoperative optimisation and it identifies patients with increased intraoperative and postoperative monitoring needs.
Risk prediction before major surgery Perioperative risk is largely determined by three domains: urgency of surgery, surgeryspecific risk factors such as invasiveness, expected blood loss or fluid shifts, and patient-specific risks such as age, comorbidities and fitness. Whereas the first domain is usually non-modifiable, the latter two are
## Strengths and limitations of this study
⇒ This study focuses on frail, elderly patients undergoing major surgery with a proven low functional capacity using cardiopulmonary exercise testing. ⇒ It implements a structured, multidisciplinary, multimodal prehabilitation intervention addressing low functional capacity, malnutrition, anaemia and smoking. ⇒ The primary endpoint is the Comprehensive Complication Index at 30 days. ⇒ Patient-reported outcomes such as Quality of Recovery are implemented as secondary outcomes. ⇒ The study is not blinded due to the nature of the intervention, however, primary outcome is assessed by a blinded adjudication committee.
Open access modifiable to some extent, offering the opportunity to adjust the surgical procedure or optimise the patient's condition prior to surgery. Several preoperative or perioperative risk scores and prediction models have been proposed and validated, such as the Portsmouth Physiological and Operative Severity Score for the enumeration of Mortality and morbidity (P-)POSSUM, Surgical Risk Score, the American College of Surgeons National Surgical Quality Improvement Programme or the Preoperative Score to Predict Postoperative Mortality for non-cardiac surgery (NCS) or the European System for Cardiac Operative Risk (EuroScore) for cardiac surgery. [bib_ref] European system for cardiac operative risk evaluation (EuroSCORE), Nashef [/bib_ref] [bib_ref] Comparison of surgical risk score, POSSUM and P-POSSUM in higher-risk surgical patients, Brooks [/bib_ref] [bib_ref] Preoperative score to predict postoperative mortality (POSPOM): derivation and validation, Manach [/bib_ref] [bib_ref] An evaluation of the POSSUM surgical scoring system, Whiteley [/bib_ref] [bib_ref] Development and evaluation of the universal ACS NSQIP surgical risk calculator: a..., Bilimoria [/bib_ref] In addition, organ-specific risk scores such as Revised Cardiac Risk Index for cardiac complications and many others have been published and widely used. Traditionally, patients' reported health state, medical history and clinical assessment were used to classify the patients' individual risk, for example, using the American Society of Anesthesiologists 12 score. Accordingly, level of fitness was reported by the patients themselves. Based on the 2022 European Society of Cardiology/European Society of Anaesthesiology (ESC/ESA) guidelines, for example, further investigations such as (cardiac) stress testing are indicated for patients with low functional capacity (less than four metabolic equivalents of task) before high-risk elective NCS and high likelihood or proven coronary artery disease or before intermediaterisk NCS when ischaemia is of concern in patients with clinical risk factors and poor functional capacity. [bib_ref] 2022 ESC guidelines on cardiovascular assessment and management of patients undergoing non-cardiac..., Halvorsen [/bib_ref] Patient-reported measures of functional capacity remain notoriously unreliable. Better performance was seen when using the Duke Activity Status Index (DASI) questionnaire, which correlated well with objective, cardiopulmonary exercise testing (CPET)-derived parameters such as peak oxygen consumption (VO2 peak ) and was able to predict cardiovascular events in a recent trial. 14 While CPET remains the gold standard for global assessment of functional capacity, it is laborious for the patient and costly. Therefore, the Canadian Cardiovascular Society recommends against performing perioperative CPET to enhance cardiac risk estimation. [bib_ref] Canadian cardiovascular Society guidelines on perioperative cardiac risk assessment and management for..., Duceppe [/bib_ref] Nevertheless, an anaerobic threshold (AT) level of <11 mL/kg/ min has been confirmed as a reliable predictor for increased perioperative risk. This also applies to the elderly patient. [bib_ref] Cardiopulmonary exercise testing as a screening test for perioperative management of major..., Older [/bib_ref] In addition, the AT has the better discriminatory power to identify patients with reduced functional capacity comparted to the 6 min walking test (6MWT), which is recommended for departments lacking CPET equipment. [bib_ref] Validity of the 6 min walk test in prediction of the anaerobic..., Sinclair [/bib_ref] Very recent evidence found that an increased VE/VCO2 slope might be an even more sophisticated prognostic marker to predict morbidity in high-risk patients having major cancer surgery or patients undergoing lobectomy for lung cancer. With increasing age, the physiological resilience to external stressors such as surgery diminishes, while the number of comorbidities rises and muscle mass decreases. In addition, elderly people are often malnourished, have a weakened immune system and impaired wound healing. All these factors contribute to a reduction in functional capacity. [bib_ref] Basic science of Frailty-Biological mechanisms of agerelated sarcopenia, Csete [/bib_ref] Despite the inconsistency of the definition of frailty, it can be assessed using a variety of tools such as the clinical frailty scale or more objective Fried frailty criteria including gait speed and handgrip strength, which are useful predictors of all-cause mortality and cardiovascular disease and death in elderly surgical and non-surgical patients. [bib_ref] Hand-grip dynamometry predicts future outcomes in aging adults, Bohannon [/bib_ref] [bib_ref] Associations of grip strength with cardiovascular, respiratory, and cancer outcomes and all..., Celis-Morales [/bib_ref] [bib_ref] Slow gait speed is associated with worse postoperative outcomes in cardiac surgery:..., Chang [/bib_ref] Similarly, standardised screening and assessments exist for the diagnosis of malnutrition and sarcopenia, which demonstrated significant overlap with frailty. [bib_ref] Nutritional screening in a cancer prehabilitation programme: a cohort study, Burden [/bib_ref] [bib_ref] Sarcopenia: revised European consensus on definition and diagnosis, Cruz-Jentoft [/bib_ref] [bib_ref] ESPEN guidelines for nutrition screening, Kondrup [/bib_ref] In summary, the elderly, frail, malnourished and sarcopenic patient is especially at risk for perioperative complications, however, many options for medical, physiological and psychological optimisation exist. [bib_ref] Recommendations for preoperative management of frailty from the Society for perioperative assessment..., Alvarez-Nebreda [/bib_ref] Interventions to reduce perioperative risk Organ specific, medically mandated interventions (as brought up by the preoperative assessment, such as treatment of relevant aortic or coronary stenosis) have to be completed prior to major surgery, irrespective of any study participation. However, despite clear guidelines on patient blood management, for example, relevant anaemia is not always corrected. In fact, less than half of Swiss hospitals had implemented a patient blood management programme in the year 2022. [bib_ref] Dissemination of patient blood management practices in Swiss intensive care units: a..., Previsdomini [/bib_ref] To empower the patient physically and psychologically to withstand the stress of surgery and to hasten recovery, the focus is widening from the intraoperative and postoperative to the perioperative period as a continuum. In addition, patients are believed to be more receptive for beneficial changes in behaviour prior to surgery than during the phase of recovery. Whereas rehabilitation is covering the postoperative period, the term prehabilitation has emerged for interventions occurring weeks before surgery.
As of today, prehabilitation is a complex approach with a combination of possible interventions (e.g., exercise, nutrition, psychosocial, and medical optimisation), and there is no precise definition of the type of intervention, the optimal dose nor duration required. This has led to widely differing interpretations and protocols of interventional trials with unimodal (addressing only one domain such as physical fitness, whereby also mixed-exercise programmes including aerobic, resistance and inspiratory muscles training were implemented) or multimodal (addressing additional domains such as anaemia, malnutrition, smoking behaviour and psychological support) interventions.
Many conflicting results for clinical effectiveness of prehabilitation interventions have been reported. [bib_ref] Prehabilitation in patients undergoing colorectal surgery fails to confer reduction in overall..., Gloor [/bib_ref] [bib_ref] Effect of multimodal prehabilitation vs postoperative rehabilitation on 30-day postoperative complications for..., Carli [/bib_ref] [bib_ref] Pre-Admission interventions (prehabilitation) to improve outcome after major elective surgery: a systematic..., Perry [/bib_ref] [bib_ref] Effect of preparative rehabilitation on recovery after cardiac surgery: a systematic review, Yau [/bib_ref] The potential for cost reduction seems plausible, but good evidence from randomised controlled trials is lacking so far. [bib_ref] Structured presurgery prehabilitation for aged patients undergoing elective surgery significantly improves surgical..., Koh [/bib_ref] [bib_ref] Costs and quality of life for prehabilitation and early rehabilitation after surgery..., Nielsen [/bib_ref] [bib_ref] A combination of enhanced recovery after surgery and prehabilitation pathways improves perioperative..., Ploussard [/bib_ref] [bib_ref] Post-Discharge impact and cost-consequence analysis of prehabilitation in high-risk patients undergoing major..., Barberan-Garcia [/bib_ref] Potential reasons for these equivocal results are several fold, with the first and most important probably being inappropriate patient selection. From eight randomised trials addressing prehabilitation before major intra-abdominal Open access cancer surgery, only two limited the study population to 'high-risk' patients, based on age, American Society of Anaesthesiologists or Dukes classification, however, none of them used objectively measured physical fitness as an inclusion criteria. [bib_ref] Prehabilitation before major intra-abdominal cancer surgery: a systematic review of randomised controlled..., Thomas [/bib_ref] Selecting patients with a relatively low risk for perioperative complications will inevitably lead to a dilution of the possible positive effects and produce negative results regardless of the intervention or surgeries studied. The small or inexistent benefit to 'lowrisk' patients does not warrant the time-demanding and resource-demanding interventions. In addition, the time required for the intervention has been found to make recruitment of large cohorts difficult. Second, poor results may have been due to implementing unimodal rather than individually tailored multimodal interventions that cover all domains of patients' health state prior to surgery, such as physical fitness, malnutrition, diabetes control, anaemia, smoking and psychological well-being.
Insufficient exercise capacity is believed to be the most important predictor for postoperative complications, [bib_ref] Prehabilitation before major abdominal surgery: a systematic review and meta-analysis, Hughes [/bib_ref] and therefore, mandates careful assessment with appropriate patient selection. While physical fitness interventions (even high-intensity/interval training) have been proven to be safe and feasible and have led to a measurably increased fitness levels (e.g., 6MWT), they failed to produce outcome benefits, possibly due to inappropriate patient selection, unimodality (e.g., lack of considerations of other factors such as malnutrition) and adherence. [bib_ref] Preoperative aerobic exercise training in elective intra-cavity surgery: a systematic review, O'doherty [/bib_ref] [bib_ref] Exercise interventions for people undergoing multimodal cancer treatment that includes surgery, Loughney [/bib_ref] [bib_ref] Effect of exercise and nutrition prehabilitation on functional capacity in esophagogastric cancer..., Minnella [/bib_ref] [bib_ref] Preoperative exercise therapy for gastrointestinal cancer patients: a systematic review, Vermillion [/bib_ref] Further, up to one-third of surgical patients are malnourished, and malnutrition has been proven to be associated with increased morbidity, mortality and cost. For malnourished patients, oral nutritional supplementation, in particular adequate protein supplementation, was shown to be effective in improving outcomes. [bib_ref] Pre-Operative nutrition support in patients undergoing gastrointestinal surgery, Burden [/bib_ref] [bib_ref] A systematic review of the cost and cost effectiveness of using standard..., Elia [/bib_ref] [bib_ref] Systematic review and metaanalysis of the effects of high protein oral nutritional..., Cawood [/bib_ref] Malnutrition is often associated with sarcopenia and frailty, making a combined intervention of exercise training and nutritional support promising. [bib_ref] Basic science of Frailty-Biological mechanisms of agerelated sarcopenia, Csete [/bib_ref] However, so far, nutritional assessment and intervention have been poorly standardised, making conclusions regarding its efficacy and interstudy comparisons difficult. [bib_ref] Current landscape of nutrition within prehabilitation oncology research: a scoping review, Gillis [/bib_ref] A further frequent metabolic comorbidity is diabetes affecting approximately 30%-40% of people undergoing elective surgery. Surgical outcomes are worse in people with diabetes with an up to three-fold higher risk of postoperative complications including poor healing, wound complications and renal dysfunction. Although inadequate glucose control was associated with poor surgical outcomes, 54 no systematic strategies to improve diabetes management prior to surgery are currently recommended and only few prehabilitation programmes included diabetes optimisation as part of a multimodal concept.
Anaemia-related perioperative blood transfusions and smoking have both been found to be related to worse outcomes and increased costs. [bib_ref] Society for perioperative assessment and quality improvement (SPAQI) consensus statement on perioperative..., Wong [/bib_ref] [bib_ref] Risks, benefits, alternatives and indications of allogenic blood transfusions, Madjdpour [/bib_ref] [bib_ref] Implementation of a patient blood management monitoring and feedback program significantly reduces..., Mehra [/bib_ref] The negative influence of preoperative anxiety or depression on postoperative outcomes such as quality of life has gained more attention. Even without an proper psychosocial intervention, physical exercise can reduce psychological distress in addition to improving cardiovascular function and ultimately improve wound healing. [bib_ref] Exercise accelerates wound healing among healthy older adults: a preliminary investigation, Emery [/bib_ref] Another factor contributing to negative results of existing multimodal prehabilitation programmes might be attributable to the simple fact that patients were randomised into the intervention group, but were not taking actively part in the programme (lack of patient adherence). [bib_ref] Effect of preoperative exercise on cardiorespiratory function and recovery after surgery: a..., Lemanu [/bib_ref] Some studies fail to demonstrate this effect by not measuring adherence rates at all [bib_ref] Prehabilitation in patients undergoing pancreaticoduodenectomy: a randomized controlled trial, Ausania [/bib_ref] So the necessity of implementing a multimodal intervention seems obvious given the important contribution of anaemia, smoking, nutritional status, physical fitness and psychological well-being. Nevertheless, even for multimodal prehabilitation, the level of evidence remains low and conflicting, with inappropriate patient selection appearing to be the main reason. In conclusion, no trials to date have limited the study population to 'high-risk' patients most prone to benefit from prehabilitation. Further, only few studies have implemented a multimodal approach targeting correction of anaemia and malnutrition, smoking cessation and improving physical fitness. There is a need for trials with stringent selection of the patients who will benefit most from preoperative optimisation and implementation of personalised comorbidity mitigation and risk factor management offered in the form of individually tailored multimodal prehabilitation. [bib_ref] Prehabilitation before surgery: is it for all patients?, West [/bib_ref] Outcome measures after major surgery The most widely accepted postoperative hard outcome measure is mortality at 30 days. [bib_ref] Comparison of surgical risk score, POSSUM and P-POSSUM in higher-risk surgical patients, Brooks [/bib_ref] Nevertheless, extending the follow-up to at least 90 days or ideally 1 year has been proposed. [bib_ref] Standards for definitions and use of outcome measures for clinical effectiveness research..., Jammer [/bib_ref] To grade postoperative complications, the Clavien-Dindo classification has been established and recommended by the ESA-European Society of Intensive Care Medicine joint taskforce on perioperative outcome measures. [bib_ref] Standards for definitions and use of outcome measures for clinical effectiveness research..., Jammer [/bib_ref] [bib_ref] Classification of surgical complications: a new proposal with evaluation in a cohort..., Dindo [/bib_ref] [bib_ref] The Clavien-Dindo classification of surgical complications: five-year experience, Clavien [/bib_ref] There is a huge variety of possible postoperative complications, and powering studies for individual complications is nearly impossible. To overcome this difficulty, the Comprehensive Complication Index (CCI) has been developed. [bib_ref] The comprehensive complication index: a novel continuous scale to measure surgical morbidity, Slankamenac [/bib_ref] It is a continuous scale to measure surgical morbidity (ranging from 0=no complications to 100=death) and can be used to reduce sample size in randomised controlled trials as it provides a more sensitive endpoint. [bib_ref] The comprehensive complication index: a novel and more sensitive endpoint for assessing..., Slankamenac [/bib_ref] [bib_ref] The comprehensive complication index CCI: a proposed modification to optimize shortterm complication..., Furrer [/bib_ref] [bib_ref] Complication reporting with the Bern comprehensive complication index CCI after open radical..., Löffel [/bib_ref] Calculation of cost savings The CCI can be used to calculate costs, as postoperative morbidity correlates highly with healthcare costs. Staiger et al found a 14% increase to the baseline cost for each 10-point increase in CCI. [bib_ref] The comprehensive complication index (CCI®) is a novel cost assessment tool for..., Staiger [/bib_ref] Therefore, a CCI reduction can serve as a surrogate marker for cost savings.
## Open access
## Study hypothesis
We hypothesise that the implementation of a multimodal, multidisciplinary prehabilitation programme over 2-4 weeks, consisting of physical exercise, nutrition, anaemia correction and smoking cessation, will result in lower CCI within 30 days after major surgery in elderly patients.
Feasibility: preliminary data After ethics approval and logistic preparations, we were able to include 18 patients from March 2022 to October 2022 [fig_ref] Table 1: Preliminary results [/fig_ref].
[formula] Female sex 2 (25%) 1 (10%) ASA score=III (n) 2 (25%) 2 (20%) ASA score ≥IV (n) 6 (75%) 8 (80%) Fried Frailty Scale (n) 1.9 (SD 1.1) 2.1 (SD 1.2) Smoking (y/n) 1 (12.5%) 0 (0%) Anaemia (y/n) 2 (25%) 4 (40%) Nutritional deficit (NRS-2002≥3) (y/n) 2 (25%) 1 (10%) [/formula]
Ischaemic heart disease or chronic heart failure (y/n) 4 (50%) 6 (60%)
[formula] Diabetes (y/n) 3 (37.5%) 3 (30%) Obesity (BMI ≥30 kg/m 2 ) (y/n) 1 (12.5%) 1 (10%) Surgical procedure [/formula]
## Open access
# Methods
## Study design
This is a national multicentre, multidisciplinary, prospective, two-arm parallel-group, randomised, controlled superiority trial with blinded outcome assessment. We aim to include 466 patients within 3 years. The present protocol complies with the Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) 2013 guidelines, defining standard protocol items for clinical trials.
## Participants and enrolment
Surgeons scheduling the operation will prescreen the patients for frailty based on the clinical frailty scale. [bib_ref] A global clinical measure of fitness and frailty in elderly people, Rockwood [/bib_ref] This tool is easy to implement in clinical practice, especially when compared with other, more sophisticated scales. [bib_ref] Accuracy and feasibility of clinically applied frailty instruments before surgery: a systematic..., Aucoin [/bib_ref] If the patient achieves at least four points on the clinical frailty scale (living with very mild frailty) and there is sufficient time for prehabilitation (at least 2 weeks), informed consent will be sought, followed by a screening CPET (baseline visit, see [fig_ref] Figure 1: Study flow chart [/fig_ref].
## Inclusion criteria
Participants are eligible if aged 65 years or over, have planned major visceral, urologic, cardiac, vascular, orthopaedic or thoracic surgery, are classified as ASA III or higher by the attending anaesthetist, and have a proven deficit in functional capacity measured during the screening CPET defined as follows: ► VO2 at AT <11 mL/kg/min or peak VO2 <14 mL/kg/ min on cycle ergometer. ► VO2 at AT <13 mL/kg/min or peak VO2 <16.5 mL/ kg/min on treadmill. ► VO2 at AT <9 mL/kg/min or peak VO2 <11.3 mL/ kg/min with arm crank. ► VE/VCO2 slope ≥33 (independent of modality).
Patients with VO2 above thresholds during the screening CPET will nevertheless be consented, in order to collect their CCI at 30 days. CCI at 30 days will be compared descriptively between patients qualifying and not qualifying for this study for qualitative clinical assessment outside the purpose of this study.
## Exclusion criteria
Patients are excluded if scheduled for emergency surgery, if unable to exercise for physiological or psychological reasons, if having cognitive disabilities making adherence to interventions impossible or if requiring dialysis due to logistic reasons.
## Preoperative assessments
All patients are assessed twice during the preoperative phase, once at inclusion (baseline) and once the day before surgery (preoperative). Measurements conducted during these visits include CPET, grip strength and maximal inspiratory pressure for the assessment of physical fitness. Bioelectrical impedance analysis 13 measurements and validated nutritional screening and assessment tools (Nutrition Risk Screening 2002 (NRS-2002) and the Subjective Global Assessment [SGA]) will be performed to quantify participants' body composition and nutritional status. Blood samples are taken to screen for anaemia (haemoglobin, reticulocyte index, renal function, iron status using transferrin saturation), nutritional and diabetic status (albumin, prealbumin, glucose, glycated haemoglobin (HbA1c)), micronutrient deficits (erythrocyte folate, holotranscobalamin, 25-hydroxy-vitamin D) and cardiac function (N-terminal-pro hormone brainderived natriuretic peptide (NT-proBNP), high-sensitivity (hs) TroponinT). For smokers, exhaled carbon monoxide (CO) is measured (Smokerlyzer Bedfont Scientific, Kent, UK) and smoking habits evaluated by questionnaire including the Fagerström test. All patients are asked to fill in the short form of the State-Trait Anxiety Inventory, [bib_ref] The comprehensive complication index (CCI®) is a novel cost assessment tool for..., Staiger [/bib_ref] the DASI and (on the day prior to operation) the Quality of Recovery (QoR-)15 questionnaire. An activity tracker (AX3, Axivity, Newcastle, UK) is provided at the screening visit and continuously worn until the preoperative visit, when it is collected from the patient.
## Group allocation (minimisation)
After completion of the screening visit (CPET), patients who fulfil the inclusion criteria are randomised using computer-generated random numbers, into either Open access intervention or control group. For group allocation, a probabilistic minimisation procedure is used to achieve balanced groups in regard to parameters known to be relevant for outcome after major surgery. We have included the following parameters in the minimisation algorithm (SecuTrial, V.6.1.1.10): type of surgery (visceral, urologic, cardiovascular, orthopaedic, thoracic), smoking, presence of anaemia (haemoglobin below 120 g/L in females, <130 g/L in males), nutritional deficit (NRS ≥3), age group (65-74, 75-84, 85+), ischaemic heart disease or heart failure, known diabetes, sex, obesity (body mass index (BMI) >30 kg/m 2 ) and use of extracorporeal circulation.
## Patient and public involvement
Patients or the public were not involved in the design, or conduct, or reporting, or dissemination plans of our research.
## Study interventions
## Control group
For the control group, there is a medical work up by the study team of the results obtained. In case of abnormalities found mandating medical interventions prior to surgery based on clinical judgement, these will be initiated either by the research team or the general practitioner (GP) at the patients' discretion. Regardless of existing deficits, the patient's GP will be informed about the patient's study participation and will receive the results of the screening assessment. All study patients wear the accelerometer between screening and preoperative visit.
## Intervention group
For patients of the intervention group, we will implement an individually tailored, multimodal prehabilitation programme consisting of physical and respiratory training, correction of nutritional deficits and anaemia and smoking cessation counselling based on the identified deficits.
## Physical activity
All patients perform a tailored exercise intervention based on their individual physical fitness level. The intervention includes 1-2 sessions (60 min each) with a licensed physical therapist trained in exercise prescription and 5 years of experience in the field of exercise prescription. The physical therapist will assess the patient's physical abilities and discuss the possibilities of incorporating at least 30 min of exercise at least five times per week at home. Based on the planned surgical intervention (e.g., hip, back), the preferences and barriers of each individual patient, the physical therapist composes a tailored personal training programme. This physical training programme is composed of endurance training (e.g., walking at moderate intensity, climbing stairs), resistance training (e.g., upper limb strengthening using a resistance band, Theraband, Akron, Ohio, USA) and inspiratory muscle training (Threshold IMT, Philips Healthcare, Horgen, Switzerland). The patient receives the personal training programme either via smartphone application or as a hard copy. The non-supervised home-based exercise training sessions will be supported by illustrative materials (smartphone application with pictures and videos or brochure with pictures). Each patient receives weekly phone calls by the physical therapist to record adherence, as a motivational strategy, to discuss any boundaries to adhering to the programme and to progress the exercises if possible. An exercise intervention might look like the following programme: Respiratory: Inspiratory muscle training (IMT) at 40% of measured maximum inspiratory pressure (MIP), 60 repetitions daily (30 morning, 30 evening); Endurance: outdoor walking at moderate intensity, 30 min 5 days/week; strength: squats with bedside support, 3×10 repetitions, as deep as pain free, 5 days/week.
## Nutritional support and management of anaemia
We aim to optimise the nutritional status to minimise the surgery-related catabolic stress. Nutritional risk will be assessed at baseline, preoperatively and postoperatively during study visits using the NRS-2002 total score. [bib_ref] Nutritional risk screening (NRS 2002): a new method based on an analysis..., Kondrup [/bib_ref] This validated screening tool attributes scores of different levels of nutritional status to patients based on weight loss, BMI and food intake, taking into account disease severity (degree of stress metabolism) and age. NRS-2002 total score is obtained by adding impaired nutritional status score (0-3 points), severity of disease score (0-3 points) and age (1 point if age ≥70 years). Patients are classified as being at high nutritional risk or malnourished (score ≥3) or at low risk (score <3), according to the total score obtained (see [fig_ref] Figure 2: Nutrition and diabetes intervention workflow [/fig_ref].
After screening with NRS-2002, nutritional status will be further quantified according to the patient generated SGA (PG-SGA) score, which defines the need for nutritional intervention: no intervention needed (score 0-1), needing education by a professional (score 2-3), requiring intervention by a certified dietitian (score [bib_ref] Identification and characterisation of the high-risk surgical population in the United Kingdom, Pearse [/bib_ref] [bib_ref] European system for cardiac operative risk evaluation (EuroSCORE), Nashef [/bib_ref] [bib_ref] Comparison of surgical risk score, POSSUM and P-POSSUM in higher-risk surgical patients, Brooks [/bib_ref] [bib_ref] Preoperative score to predict postoperative mortality (POSPOM): derivation and validation, Manach [/bib_ref] [bib_ref] An evaluation of the POSSUM surgical scoring system, Whiteley [/bib_ref] and having a critical need for symptom management and/or nutrient intervention (score ≥9). [bib_ref] Definition of standardized nutritional assessment and interventional pathways in oncology, Ottery [/bib_ref] Patients with low risk for malnutrition (NRS-2002 <3) will be provided with dietary advice on how to achieve set individual nutritional targets (energy intake according to the Harris-Benedict equation and protein intake of >1 g/kg BW/day). Adjusted bodyweight will be used in case of BMI >25 to prevent overestimation of nutritional requirements.
Patients at high risk for being malnourished (NRS-2002 ≥3, PG-SGA ≥4) will receive dietary counselling and individualised plans to meet their energy and protein requirements. Individual energy needs will be calculated using the Harris-Benedict equation. A protein intake of 1.2-1.5 g/ kg BW will be recommended to all malnourished participants (using adjusted body weight when BMI >25, as described above). To facilitate the achievement of these goals, oral nutritional supplements will be prescribed. The aim is to achieve at least 75% of the set energy and protein goal. The adherence in the malnourished group Open access will be checked by performing 24 hours dietary recall 3 days after baseline visit. In case of reaching <75% of set dietary goals, the intervention will be escalated to enteral nutrition. If the goals are not fulfilled during the following 3 days as evaluated with 24 hours dietary recall, the intervention will be escalated to parenteral nutrition.
If scheduled for major abdominal surgery and if indication exists according to Swiss Society for Clinical Nutrition, patients will additionally receive immunonutrition (e.g., high protein oral supplements enriched with immunonutrients such as arginine, omega-3-fatty acids and nucleotides) for five consecutive days before surgery, as per ERAS guidelines. All participants will receive two doses of carbohydrate loading, one dose on the evening before and one up to 2 hours prior to surgery. [bib_ref] ESPEN practical guideline: clinical nutrition in surgery, Weimann [/bib_ref] In patients with an established or newly diagnosed diabetes and HbA1c >7.5%, glucose levels will be optimised using pre-scribed glucose monitoring and treatment intensification where appropriate. Delivered strategies represent best practice of diabetes care based on the European Association for the Study of Diabetes (EASD)/American Diabetes Association (ADA) position statement. [bib_ref] Management of hyperglycaemia in type 2 diabetes, 2015: a patient-centred approach. update..., Inzucchi [/bib_ref] Independent of the nutritional status, individual deficits in vitamin D, iron, vitamin B 12 and folate will be addressed. Vitamin D is associated with muscle mass and muscle strength. Additionally, hypovitaminosis D was found to be correlated with increased risk of postoperative complications. It will be supplemented daily according to the blood sample values: 1000 U/day for patients with vitamin D deficiency (25-hydroxy-vitamin D <50 nmol/L) and a one-off substitution of 150 000 U for patients with severe vitamin D deficiency (25-hydroxy-vitamin D <25 nmol/L).In case of severe renal anaemia (haemoglobin <100 g/L, estimated glomerular filtration rate <45 mL/min/1.73 m 2 and a reticulocyte index below 2), a one-off erythropoietin application (Mircera, Vifor Pharma, Switzerland) will be provided.
## Smoking cessation
The smoking cessation programme includes intensive counselling by a graduate psychology student during three phone-call sessions as well as dispensary of nicotine replacement therapy (NRT) perioperatively if desired by the patient. NRT consists of transdermal application for moderate to heavy smokers and individual supplementation with chewing gum or lozenges (Nicotinell, GlaxoSmithKline, Brentford, UK) according to the current guidelines. NRT will either be handed out during the nutritional intervention session or sent via postal mail. The goal is to achieve a reduction in smoking quantity (cigarettes per day) of 80% before surgery.
## Compliance with the study interventions
Adherence rates to the physical fitness intervention are primarily collected by patient-recorded standardised physical activity log books, by weekly phone calls and complemented by accelerometer data (AX3, Axivity, Newcastle, UK). Successful completion of the exercise programme is defined as adherence to 80% of prescribed exercise sessions.
For patients with an NRS-2002 ≥3 or PG-SGA ≥4, weekly phone calls by the dietician will secure adherence to the study intervention. In addition, the patients will document the intake of prescribed supplements in a log book.
The three phone call smoking cessation counselling sessions for smokers are intended to monitor and further adherence to smoking cessation. Further, exhaled CO is measured at every visit.
Reasons for non-compliance are recorded during all phone calls and strategies to increase compliance are sought in the discussion with the patient.
## Perioperative management
Perioperative management follows the usual care of the corresponding clinic, usually following standardised, multielement ERAS guidelines. Management decisions such as extension of monitoring and admission to postoperative care (postoperative care unit vs intermediate or intensive care unit (ICU)) are purely based on clinical judgement, taking the results of the preoperative testing into account as it is accessible by the clinicians through the electronic healthcare record system.
In case of raised preoperative NTproBNP >200 pg/mL, hsTroponinT will be measured 6 hours after surgery as well as on the first and second postoperative day. [bib_ref] Preoperative N-terminal pro-Btype natriuretic peptide and cardiovascular events after noncardiac surgery: a..., Duceppe [/bib_ref] Postoperative assessment The postoperative assessment as described above will be repeated at 30 days after surgery when the patient is scheduled for surgical follow-up. The only difference from the preoperative assessment is that micronutrient and iron status are not measured again.
Blinding procedure This is an unblinded study due to the nature of the intervention. Nevertheless, the treating anaesthetist, while having access to the results of the preoperative assessment results via EHR, is blinded to group allocation. Primary end point adjudication committee will also be blinded (see below).
## Data collection
All data will be collected by the research team and documented in a research database (RedCap) according to local legislation. Data will be verified by dedicated members of the research team. Original source data is available in the EHR or in patients' files.
## End point adjudication committee
In order to standardise the review process and to reduce bias and variability in assessing the primary outcome, the CCI at 30 days will be assessed by an independent and blinded committee of three experts, which are otherwise not involved in the trial. The experts are yet to be nominated.
## Open access
## Data monitoring
The data will be monitored by an external institution (Clinical Trials Unit, University of Bern) at regular intervals according to a preset monitoring plan to ensure the safety of the trial participants and the integrity of the trial data.
## Primary endpoint
Postoperative complications at 30 days after surgery scored by the CCI, assessed by a blinded, independent expert committee.
## Secondary endpoints
Secondary outcomes are measured before prehabilitation (screening visit) and thereafter (preoperative visit) and at 30 days after surgery (postoperative visit) unless otherwise specified:
[formula] Cardiovascular [/formula]
## Sample size
Based on internal data (Bern University Hospital), median CCI is expected to be 30 (IQR [bib_ref] The Marsden morbidity index: the derivation and validation of a simple risk..., Nawoor-Quinn [/bib_ref] [bib_ref] Basic science of Frailty-Biological mechanisms of agerelated sarcopenia, Csete [/bib_ref] [bib_ref] Hand-grip dynamometry predicts future outcomes in aging adults, Bohannon [/bib_ref] [bib_ref] Associations of grip strength with cardiovascular, respiratory, and cancer outcomes and all..., Celis-Morales [/bib_ref] [bib_ref] Slow gait speed is associated with worse postoperative outcomes in cardiac surgery:..., Chang [/bib_ref] [bib_ref] Nutritional screening in a cancer prehabilitation programme: a cohort study, Burden [/bib_ref] [bib_ref] Sarcopenia: revised European consensus on definition and diagnosis, Cruz-Jentoft [/bib_ref] [bib_ref] ESPEN guidelines for nutrition screening, Kondrup [/bib_ref] [bib_ref] Recommendations for preoperative management of frailty from the Society for perioperative assessment..., Alvarez-Nebreda [/bib_ref] [bib_ref] Dissemination of patient blood management practices in Swiss intensive care units: a..., Previsdomini [/bib_ref] [bib_ref] Prehabilitation in patients undergoing colorectal surgery fails to confer reduction in overall..., Gloor [/bib_ref] [bib_ref] Effect of multimodal prehabilitation vs postoperative rehabilitation on 30-day postoperative complications for..., Carli [/bib_ref] [bib_ref] Pre-Admission interventions (prehabilitation) to improve outcome after major elective surgery: a systematic..., Perry [/bib_ref] [bib_ref] Effect of preparative rehabilitation on recovery after cardiac surgery: a systematic review, Yau [/bib_ref] [bib_ref] Structured presurgery prehabilitation for aged patients undergoing elective surgery significantly improves surgical..., Koh [/bib_ref] [bib_ref] Costs and quality of life for prehabilitation and early rehabilitation after surgery..., Nielsen [/bib_ref] [bib_ref] A combination of enhanced recovery after surgery and prehabilitation pathways improves perioperative..., Ploussard [/bib_ref]. Assuming an average clinically relevant improvement of 15% in the intervention group compared with the control group, the sample size calculation for a non-parametric Wilcoxon-Mann-Whitney test resulted in a total patient sample of 388 (assuming a significance level alpha of 0.05 and power of 0.80). We expect a dropout rate of 20%. We, therefore, need 466 patients: 233 in each arm. We aim to complete the inclusion within 3 years.
# Statistical analysis
Data will be analysed on an intention-to-treat basis (primary outcome). Primary and secondary outcomes for the intervention and control groups will be compared. Primary outcome: We expect that CCI will be skewed, so we will describe CCI as the median plus first and third quartiles. To test the hypothesis (H0) that the study arms result in similar CCIs (in other words, prehabilitation does not prevent postoperative complications), we will use a generalised linear model that can account for skewed data and zero inflation. The model will be adjusted for the minimisation factors used at randomisation. If no adequate model can be fit, the non-parametric Mann-Whitney U test will be used.
Secondary outcomes: All secondary outcomes will be described as means plus SD or median plus first and third quartiles for continuous data as appropriate. Categorical parameters will be described as frequencies plus percentage per time point. To account for repeat measurements of patients, we will use generalised linear mixed models to test the hypothesis of both study arms being equal in terms of secondary outcomes over time. Models will be adjusted for minimisation factors as well as baseline values (before randomisation) of the respective outcome.
Data distribution will be assessed by visual inspection of Q-Q plots.
As a secondary analysis, we will perform a PP analysis. Since the time between randomisation and surgery might differ between the two randomisation groups, we will additionally adjust generalised linear models of the primary outcome for this factor in a sensitivity analysis. Further, we will analyse the impact of adherence to the interventions to the primary outcome.
# Subgroup analysis
We will study whether effects differ between subgroups. Subgroup analyses will be performed according to the minimisation parameters (ie, surgery type, anaemia, Open access nutritional deficit, age, frailty score, ischaemic heart disease/heart failure).
## Ethics and dissemination
The trial will be conducted according the current version of the Declaration of Helsinki, International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) Guideline for Good Clinical Practice (GCP), and applicable national regulations, that is, the Human Research Act in Switzerland and has been accepted by the responsible Ethics Committee (REC) of the Canton of Berne, Switzerland (Kantonale Ethikkomission Bern 2020-01690). After initial ethics approval, subsequent protocol amendments will be submitted to the REC. Written informed consent will be obtained from all participants before entering the study.
Meetings and project presentations will be performed at the involved surgical clinics to advertise our project and raise awareness in order to motivate recruitment at regular intervals.
Results of the trial are expected to be published in major medical journals and to be presented at international congresses.
Authorship for the project will be based on the Uniform Requirements for Manuscripts of the International Committee of Medical Journal Editors (http:// www.icmje.org/#author). We followed the SPIRIT checklist when writing our report. [bib_ref] Spirit 2013 explanation and elaboration: guidance for protocols of clinical trials, Chan [/bib_ref] Public access to the dataset on patient-level is not planned but possible on reasonable request.
[fig] Figure 1: Study flow chart. CFS, Clinical Frailty Scale; CPET, cardiopulmonary exercise testing. [/fig]
[fig] Figure 2: Nutrition and diabetes intervention workflow. NRS-2002, Nutrition Risk Screening 2002; PG-SGA, The Scored Patient Generated Subjective Global Assessment; 25-OH-D, 25-hydroxy-vitamin D; RBC-Folate, Erythrocyte Folate; Hb, Haemoglobin; eGFR, estimated Glomerular Filtration Rate; EPO, erythropoietin; HbA1c, glycated haemoglobin; HBE, Harris-Benedict Equation; PAL, Physical Activity Level; BW, bodyweight; ON, Oral Nutrition; ONS, Oral Nutritional Supplements; EN, Enteral Nutrition. * Usage of adjusted bodyweight, if BMI>25. [/fig]
[table] Table 1: Preliminary results [/table]
|
Brugada syndrome and sinus node dysfunction
Brugada syndrome (BrS) is a well-known catastrophic disease first reported in 1992 by the Brugada brothers. Ventricular fibrillation (VF) is an essential arrhythmia inBrS. An association between BrS and atrial tachyarrhythmias is not uncommon.However, sinus node dysfunction (SND) associated with BrS has not been well discussed. In this review, we focus on the association between BrS and SND. Based on previous reports describing clinical, epidemiological, and genetic evidence, SND is not a rare concomitant disorder in BrS. BrS may be a multiple conduction or arrhythmogenic disorder including not only the His-Purkinje system and right ventricle, but also the sinus node and atrium, derived from ion channel mutations.K E Y W O R D SBrugada syndrome, overlap syndrome, sick sinus syndrome, sinus node dysfunction, sodium channel mutationThis is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.
# | introduction
Brugada syndrome (BrS) is a well-known catastrophic disease reported in 1992 by the brother cardiologists Pedro and Josep Brugada. [bib_ref] Right bundle branch block, persistent ST segment elevation and sudden cardiac death:..., Brugada [/bib_ref] Although ventricular fibrillation (VF) is an essential arrhythmia in BrS, an association between BrS and atrial tachyarrhythmias is not uncommon. Atrial fibrillation (AF) is the most common arrhythmia other than VF in BrS, and AF is well known as a major cause of inappropriate shock from an implantable cardioverter defibrillator (ICD). [bib_ref] Atrial fibrillation and Brugada syndrome, Francis [/bib_ref] However, sinus node dysfunction (SND) associated with BrS has not been well discussed. In this review, we focus on the association between BrS and SND.
## | clinical case reports of
## Brugada synd rome associated with sinus node dysfunction
Twenty-four years ago, we treated a patient with idiopathic VF with incomplete right bundle branch block and persistent ST elevation in the right precordial leads, which was later named "Brugada syndrome," the case report was published in 1993. [bib_ref] A case of idiopathic ventricular fibrillation with incomplete right bundle branch block..., Sumiyoshi [/bib_ref] One year ago, this patient was diagnosed as having sick sinus syndrome (SSS), and a cardiac pacemaker implanted . [bib_ref] Sinus node dysfunction concomitant with Brugada syndrome, Sumiyoshi [/bib_ref] In 1993, we thought that the combination of BrS and SND occurred incidentally, and we had never imagined a genetic correlation between the two diseases.
We later reported three adult patients with BrS and SND in 2005 [bib_ref] Sinus node dysfunction concomitant with Brugada syndrome, Sumiyoshi [/bib_ref] : a 42-year-old man (the same case as 3 ), a 62-year-old man, and a 49-year-old woman (the same case as 5 ). Spontaneous sinus pause > 3 sec was observed clinically in all three patients, and a prolonged sinus node (SN) recovery time was observed during the electrophysiological study (EPS) in two of the patients. In all three patients, SND was recognized before an episode of VF. Two of the patients had a pacemaker implanted before the ICD implantation.
There have been several case reports of BrS associated with SND or SSS since 2005 as shown in . When he was a teenager, he experienced syncope, and VF was induced by an EPS. When he was in his twenties, pilsicainideinduced type 1 ECG was documented. His pacemaker was then upgraded to an ICD.
The clinical course in two of the three young patients described above (ie, those in the Shimizu and Nakajima reports) is very interesting because BrS tends to become obvious after the diagnosis of SSS is made. The popularization of the challenge test using a sodium channel blocker may be one reason. In addition, the frequency of Brugada-type (type 1) ECG among adults has been reported as 0.05%-0.28%, [bib_ref] A survey in the incidence of right bundle branch block with ST..., Tohyou [/bib_ref] [bib_ref] Prevalence of the Brugada syndrome in an apparently healthy population, Hermida [/bib_ref] [bib_ref] The prevalence, incidence and prognostic value of the Brugada-type electrocardiogram: a population-based..., Matsuo [/bib_ref] [bib_ref] Prevalence and mortality of the Brugada-type electrocardiogram in one city in Japan, Miyasaka [/bib_ref] [bib_ref] Prevalence of asymptomatic ST segment elevation in right precordial leads with right..., Furuhashi [/bib_ref] [bib_ref] Proportion and prognosis of healthy people with coved or saddle-back type ST..., Sasabe [/bib_ref] which is higher than that among children
## 4:57 pm
F I G U R E 1 A 42-year-old man with Brugada syndrome associated with sinus node dysfunction. Twelve-lead ECG just before ventricular fibrillation (VF) shows the coved-type ST elevation in leads V1-3 (top). ECG monitoring shows a sinus pause of 3.6 s in the evening (middle). Spontaneous VF occurred after midnight (bottom) and was successfully converted to sinus rhythm by external DC shock. Modified with permission from 4 carriers. Therefore, in some juvenile SSS patients, the phenotype of BrS may become apparent in adulthood.
## | prevalence of the association between brugada syndrome and sinus node dysfunction
The relationship between BrS and SND was first reported by Morita et al [bib_ref] Sinus node function in patients with Brugada-type ECG, Morita [/bib_ref] [bib_ref] Drug-induced Brugada syndrome in children. Clinical features, device-based management, and longterm follow-up, Conte [/bib_ref] [bib_ref] Asymptomatic Brugada syndrome. Clinical characterization and long-term prognosis, Sieira [/bib_ref] [bib_ref] Clinical characterization and longterm prognosis of women with Brugada syndrome, Sieira [/bib_ref] In children, the prevalence of SND seems to be higher (6.7%-9.0%) compared to the adult patients. [bib_ref] Drug-induced Brugada syndrome in children. Clinical features, device-based management, and longterm follow-up, Conte [/bib_ref] [bib_ref] Clinical aspects and prognosis of Brugada syndrome in children, Probst [/bib_ref] [bib_ref] A clinical score model to predict lethal events in young patients (≤19..., Gonzalez Corcia [/bib_ref] Conversely, we studied the prevalence of Brugada-type ECG in 487 consecutive patients who had been diagnosed as having SSS and were implanted with a cardiac pacemaker, [bib_ref] Prevalence of the Brugada-type electrocardiogram and incidence of Brugada syndrome in patients..., Hayashi [/bib_ref] and our analyses demonstrated that the prevalence of Brugada-type ECG in this SSS group was higher (0.82%) compared to the general population (0.005%-0.28%). [bib_ref] A survey in the incidence of right bundle branch block with ST..., Tohyou [/bib_ref] [bib_ref] Prevalence of the Brugada syndrome in an apparently healthy population, Hermida [/bib_ref] [bib_ref] The prevalence, incidence and prognostic value of the Brugada-type electrocardiogram: a population-based..., Matsuo [/bib_ref] [bib_ref] Prevalence and mortality of the Brugada-type electrocardiogram in one city in Japan, Miyasaka [/bib_ref] [bib_ref] Prevalence of asymptomatic ST segment elevation in right precordial leads with right..., Furuhashi [/bib_ref] [bib_ref] Proportion and prognosis of healthy people with coved or saddle-back type ST..., Sasabe [/bib_ref] [bib_ref] Prevalence of right bundle branch block and right precordial ST-segment elevation (Brugadatype..., Yamakawa [/bib_ref] [bib_ref] Prevalence and time of appearance of Brugada electrocardiographic pattern in young male..., Yoshinaga [/bib_ref] [bib_ref] Prevalence and clinical course of the juveniles with Brugada-type ECG in Japanese..., Oe [/bib_ref] Thus, SND seems to be not uncommon among individuals with BrS.
## | ge netic association between
## Brugada syndrome and sinus node dysfunction
In 1998, Chen et al 32 reported the first mutation linked to BrS in the sodium channel gene SCN5A. To date, 20 subtypes of gene mutation responsible for BrS have been identified. 33 SCA5A mutations are the most common type, found in 20%-30% of BrS probands. [bib_ref] Genetics of Brugada syndrome, Watanabe [/bib_ref] SND has also been reported in patients with a sodium channel mutation. [bib_ref] A sodium-channel mutation causes isolated cardiac conduction disease, Tan [/bib_ref] [bib_ref] Congenital sick sinus syndrome caused by recessive mutations in the cardiac sodium..., Benson [/bib_ref] Prolongation of the action potential of SN cells and slowing of diastolic depolarization due to an abnormal sodium channel gene may contribute to SND. [bib_ref] Possible bradycardic mode of death and successful pacemaker treatment in a large..., Van Den Berg [/bib_ref] In addition, reduced sodium current could account for both BrS and a conduction disturbance in the sinoatrial region. [bib_ref] Long QT syndrome, Brugada syndrome, and conduction system disease are linked to..., Grant [/bib_ref] A possible bradycardic mode of death in carriers of the mutant SCN5A gene in a family with long QT syndrome (LQT) and BrS was proposed by van den Berg et al. [bib_ref] Possible bradycardic mode of death and successful pacemaker treatment in a large..., Van Den Berg [/bib_ref] They showed that one carrier who had a mutation of SCN5A (1795insD) suffered from sinus arrest > 9 second. A single mutation in SCN5A has been reported to be able to cause both the BrS and SSS phenotypes. [bib_ref] High risk for bradyarrhythmic complications in patients with Brugada syndrome caused be..., Makiyama [/bib_ref] [bib_ref] Long QT syndrome, Brugada syndrome, and conduction system disease are linked to..., Grant [/bib_ref] [bib_ref] A mutation in the human cardiac sodium channel (E161K) contributes to sick..., Smits [/bib_ref] Makiyama et al [bib_ref] High risk for bradyarrhythmic complications in patients with Brugada syndrome caused be..., Makiyama [/bib_ref] [bib_ref] Common variants at SCN5A-SCN10A and HEY2 are associated with Brugada syndrome, a..., Bezzina [/bib_ref] Yagihara et al [bib_ref] Variants in the SCN5A promoter associated with various arrhythmia phenotypes, Yagihara [/bib_ref]
## | prognostic value of sinus node
## Dysfunction in patients with brugada syndrome as a possible risk factor
In patients with BrS, SND was reported to be a risk factor significantly associated with future lethal arrhythmic events: sudden cardiac death (SCD), aborted SCD, or ICD shock. [bib_ref] Asymptomatic Brugada syndrome. Clinical characterization and long-term prognosis, Sieira [/bib_ref] only the His-Purkinje system and right ventricle, but also the SN and atrium, derived from ion channel mutations.
## Conf lict of i nterest
Authors declare no conflict of interests for this article.
## O r c i d
## Masataka
Sumiyoshi http://orcid.org/0000-0002-2644-6356
[fig] 0: 005%-0.06%).12,[18][19][20] The age of phenotype manifestation has been reported to be different between SSS and BrS in family members with SCN5A mutations. Abe et al21 reported that the mean age of the SSS manifestation in individuals with SCN5A mutation was considerably less (20 AE 3.4 years) than the mean age of those affected with BrS, which typically manifests during adulthood at a mean age of around 40 years. Abe et al 21 suggested that SND is the earliest electrophysiological manifestation of SCN5A mutation [/fig]
[table] Table 2: shows the prevalence of the combination with BrS and SND in the previous studies. Bordachar et al 23 demonstrated a high prevalence of SND, in 10 (17%) of 59 patients with BrS. However, that high prevalence was derived from the EPS-induced SND cases, not the clinically documented SND or SSS cases. In adults, although the prevalence of clinically documented SND in patients with BrS was relatively high (7.9% and 8.8%) in the small-population studies, 24,25 it was not so high (1.1%-1.7%) in the more recent large-population studies. [/table]
|
Prosocial Orientation Alters Network Dynamics and Fosters Cooperation
Dynamic networks have been shown to increase cooperation, but prior findings are compatible with two different mechanisms for the evolution of cooperation. It may be that dynamic networks promote cooperation even in networks composed entirely of egoists, who strategically cooperate to attract and maintain profitable interaction partners. Alternatively, drawing on recent insights into heterogeneous social preferences, we expect that dynamic networks will increase cooperation only when nodes are occupied by persons with more prosocial preferences, who tend to attract and keep more cooperative partners relative to egoists. Our experiment used a standard procedure to classify participants a priori as egoistic or prosocial and then embedded them in homogeneous networks of all prosocials or all egoists, or in heterogeneous networks (50/50). Participants then interacted in repeated prisoner's dilemma games with alters in both static and dynamic networks. In both heterogeneous and homogeneous networks, we find dynamic networks only promote cooperation among prosocials. Resulting from their greater cooperation, prosocials' relations are more stable, yielding substantially higher fitness compared to egoists in both heterogeneous and homogeneous dynamic networks. Our results suggest that a key to the evolution and stability of cooperation is the ability of those with prosocial preferences to alter their networks.Published: xx xx xxxx OPEN www.nature.com/scientificreports/ 2 Scientific RepoRts | 7: 357 |
Understanding the conditions that promote other-regarding motivations and cooperative behaviors are fundamental puzzles for the social and biological sciences [bib_ref] Five rules for the evolution of cooperation, Nowak [/bib_ref] [bib_ref] Social dilemmas: The anatomy of cooperation, Kollock [/bib_ref] [bib_ref] Human cooperation, Rand [/bib_ref] [bib_ref] The evolution of trust and cooperation between strangers: A computational model, Macy [/bib_ref] [bib_ref] Beyond altruism: Sociological foundations of cooperation and prosocial behavior, Simpson [/bib_ref]. Recent work has focused on structured or networked population as a key mechanism in the evolution of cooperation. In particular, dynamic networks, or networks where ties can be formed and/or broken, generate cooperation by allowing individuals to shed ties to non-cooperators or defectors [bib_ref] Strong ties promote the evolution of cooperation in dynamic networks, Melamed [/bib_ref] [bib_ref] Dynamic social networks promote cooperation in experiments with humans, Rand [/bib_ref] [bib_ref] Cooperation and assortativity with dynamic partner updating, Wang [/bib_ref] [bib_ref] Co-evolution of behaviour and social network structure promotes human cooperation, Fehl [/bib_ref] [bib_ref] Coevolution of strategy and structure in complex networks with dynamical linking, Pacheco [/bib_ref] [bib_ref] Evolutionary dynamics of social dilemmas in structured heterogeneous populations, Santos [/bib_ref]. In turn, defectors may alter their strategies to retain their remaining ties. In these networks, cooperation diminishes through time [bib_ref] Cooperation and assortativity with dynamic partner updating, Wang [/bib_ref] , but remains a more viable strategy than in static networks.
Existing findings are compatible with two different explanations of why and when dynamic networks promote cooperation. First, dynamic networks might increase cooperation even in networks composed entirely of egoists, who strategically cooperate in order to attract and maintain profitable interaction partners [bib_ref] Reputation and efficiency in social interactions: An example of network effects, Raub [/bib_ref] [bib_ref] Cooperation prevails when individuals adjust their social ties, Santos [/bib_ref]. But we expected that dynamic networks increase cooperation mainly when nodes are occupied by those with more prosocial preferences [bib_ref] Development of prosocial, individualistic, and competitive orientations: theory and preliminary evidence, Van Lange [/bib_ref] [bib_ref] Altruistic punishment in humans, Fehr [/bib_ref] [bib_ref] Social value orientation and cooperation in social dilemmas: A meta-analysis, Balliet [/bib_ref] : because they are more cooperative, prosocials [bib_ref] The pursuit of joint outcomes and equality in outcomes: An integrative model..., Van Lange [/bib_ref] [bib_ref] Social value orientation and cooperation in social dilemmas: A review and conceptual..., Bogaert [/bib_ref] will tend to attract and keep more cooperative partners relative to those with egoistic preferences. These two accounts have very different implications for the evolution of prosocial preferences and behaviors. But prior work on dynamic networks ignores insights from the literature on social values, either assuming that all actors are egoistic, or inferring "types" (prosocial versus egoistic) from the behaviors that those dispositions are supposed to explain (cooperation versus non-cooperation). As a consequence, previous work cannot discriminate between the two accounts of when and why dynamic networks promote cooperation.
Our experiments included a total of 360 participants interacting in twelve-person networks. Before the experiment took place (average of 26.4 days), we measured prosocial versus egoistic dispositions a priori using a standard procedure for measuring social values [bib_ref] The pursuit of joint outcomes and equality in outcomes: An integrative model..., Van Lange [/bib_ref] [bib_ref] The virtues of gossip: reputational information sharing as prosocial behavior, Feinberg [/bib_ref]. We used these measures to sort participants into homogeneous networks of all prosocials or all egoists, or heterogeneous networks, comprised of 50% prosocials and 50% egoists. Each network of 12 completed both a static and dynamic condition, in random order, for a total of 60 networks (see SI Appendix). Network ties represented an opportunity to interact in a prisoner's dilemma (PD). We predicted that prosocials would keep their ties longer as a result of their higher levels of cooperation, which would result in increased earnings. Moreover, in dynamic networks, we expected greater inequality in heterogeneous (versus homogeneous) networks, with that inequality favoring prosocials.
For both the static and dynamic phases, there were 12 rounds of interaction in the PD that entailed choosing whether to cooperate or defect with each alter. Participants were not told how many rounds there would be. Prior studies typically force participants to either cooperate or defect with all of their alters [bib_ref] Dynamic social networks promote cooperation in experiments with humans, Rand [/bib_ref] [bib_ref] Cooperation and assortativity with dynamic partner updating, Wang [/bib_ref] [bib_ref] Quality versus quantity of social ties in experimental cooperative networks, Shirado [/bib_ref] , but in the interest of realism and consistent with findings that participants use conditional responses in PD games [bib_ref] Are people conditionally cooperative? Evidence from a public goods experiment, Fischbacher [/bib_ref] [bib_ref] Individual differences in the game motives of own, relative, and joint gain, Kuhlman [/bib_ref] [bib_ref] Elements of reciprocity and social value orientation, Parks [/bib_ref] , we allowed them to make independent decisions for each relation. The incentive structure for the PD followed similar studies [bib_ref] Cooperation and assortativity with dynamic partner updating, Wang [/bib_ref]. Mutual cooperation resulted in +4 points each; mutual defection resulted in +1 point each; and unilateral defection and unilateral cooperation resulted in +7 and −1 points each, respectively.
Each participant initially had three ties. In static networks, these ties did not change. In dynamic networks, participants were asked whether they wanted to drop an alter after each round. If so, they were able to pick which tie to unilaterally delete. Following prior work, ties were not replaced for participants who were dropped [bib_ref] Strong ties promote the evolution of cooperation in dynamic networks, Melamed [/bib_ref] [bib_ref] Dynamic social networks promote cooperation in experiments with humans, Rand [/bib_ref] [bib_ref] Quality versus quantity of social ties in experimental cooperative networks, Shirado [/bib_ref]. Participants who dropped an alter were given a new tie to a random new alter with whom they had not previously interacted. In principle, new ties can be formed based on reputations [bib_ref] Biological markets and the effects of partner choice on cooperation and friendship, Barclay [/bib_ref] , simply at random, or at random from some subpopulation [bib_ref] Co-evolution of behaviour and social network structure promotes human cooperation, Fehl [/bib_ref]. Prior work on dynamic networks [bib_ref] Dynamic social networks promote cooperation in experiments with humans, Rand [/bib_ref] [bib_ref] Cooperation and assortativity with dynamic partner updating, Wang [/bib_ref] [bib_ref] Quality versus quantity of social ties in experimental cooperative networks, Shirado [/bib_ref] gives participants seeking new ties reputational information on prospective alters. But here we sought to isolate the effects of network dynamics by not giving participants any reputational information on prospective partners. Either of the random mechanisms would allow us isolate the effects of network dynamics, but we limited connections to those who had not previously interacted. The fact that participants could not reconnect to previously severed ties (or to those who had severed ties to them) is common in the real-world. For instance, we do not typically remarry those we have divorced; similarly, we do not generally reestablish business partnerships we have severed due to the partner's untrustworthiness, etc.
# Results
Among prosocials, dynamic networks promote cooperation in both homogeneous networks (b = 0.98, p < 0.01) and heterogeneous networks (b = 1.01, p < 0.05; unless otherwise noted, all p-values determined using (generalized) linear mixed models accounting for the nested structure of the data (SI Appendix)). Among egoists, the effect of dynamic networks is not significantly different from zero in either homogeneous (b = 0.65, p = n.s.) or heterogeneous networks (b = 0.603, p = n.s.). Only when comparing homogeneous static to heterogeneous dynamic networks do we find an increase in cooperation among egoists (b = 0.96, p < 0.05).
To show how these processes vary with time, [fig_ref] Figure 1: Marginal probability of cooperation with each alter in [/fig_ref] and B plot the marginal probabilities of cooperating across the 12 rounds in the homogeneous (1A) and heterogeneous (1B) networks. Beginning with the homogeneous networks, Round 1 shows no differences in cooperation rates. But even in round 2, prosocials in homogeneous dynamic networks cooperate at higher levels than all other groups (p < 0.05). Further, whereas all other groups exhibit the characteristic decline in cooperation through time 3 , prosocials in homogeneous dynamic networks cooperate at rates heretofore unseen in this literature. [fig_ref] Figure 1: Marginal probability of cooperation with each alter in [/fig_ref] shows similar patterns for heterogeneous networks, except that the mixing reduces cooperation among prosocials and increases cooperation among egoists, yielding less pronounced differences between the two. Together these findings suggest that whether dynamic networks promote cooperation depends crucially on the presence of prosocials.
A conditional logistic regression shows that participants were less likely to drop alters who cooperated (15%) over those who defected (41%, p < 0.001). As prosocials are more cooperative, this translates into more durable relationships between them. In both homogeneous and heterogeneous networks, prosocials' relationships lasted an average of almost one round longer (b = 0.97, p < 0.001) than egoists' relationships. Importantly, we find that the number of times participants cooperated with each alter explains the difference between egoists' and prosocials' relationship duration. Each time participants cooperated with an alter, that relationship lasted 0.91 rounds longer (p < 0.001), on average, and when this term is included, the effect of participants' prosociality becomes indistinguishable from zero (b = −0.26, p = n.s.).
One implication of the greater stability of prosocials' ties is that their networks become more homophilous 26-28 over time, as shown in [fig_ref] Figure 2: Marginal [/fig_ref]. Specifically, prosocials' networks were 15.6% more homophilous by the end of the experiment (p < 0.001). Recall that those who dropped alters were randomly connected to new alters, whereas the ties of those who were dropped by others were not replaced. We found that prosocials acquired more ties than egoists in heterogeneous dynamic networks, as shown in [fig_ref] Figure 2: Marginal [/fig_ref]. By the end of the study, egoists had lost an average of 0.36 ties, whereas prosocials had gained an average of 0.48 ties (both p < 0.01). These findings on homophily and number of ties are striking given that participants were not allowed to select their partners based, e.g., on reputations. Instead, partners were assigned at random. Thus, homophily and tie frequency emerged simply as a result of selective shedding of unwanted ties.
We now assess how the outcomes reported thus far impact earnings. Dynamic networks do not significantly increase the earnings of egoists in either homogeneous (b = 0.97, p = n.s.) or heterogeneous (b = 0.89, p = n.s.) networks. By contrast, prosocials in dynamic networks -either homogeneous (b = 3.96, p < 0.001) or heterogeneous (b = 2.18, p < 0.001) -earn substantially more. [fig_ref] Figure 3: Marginal earnings/fitness in [/fig_ref] and B show marginal earnings over time. Mediation analyses demonstrate that the average duration of relationships in a given round explains the effect of prosociality on earnings in both homogeneous and heterogeneous dynamic networks. For each round increase in average relation duration, prosocials in dynamic networks earn 0.46 additional points (p < 0.001), while the effect of duration is insignificant for egoists (b = 0.11, p = n.s.).
Following recent work 29 , we also investigated how different networks lead to inequality in earnings over time. Based on the patterns reported thus far, we expected that, in heterogeneous populations, dynamic networks would promote greater inequality, favoring prosocials. [fig_ref] Figure 4: Marginal inequality [/fig_ref] gives marginal inequality (Gini coefficients) at the network level for the heterogeneous dynamic groups. As expected, inequality increases in the dynamic networks, but remains stable in static networks. Mediation analyses show that the increased earnings of prosocials in the dynamic networks explain the effect of network structure (static or dynamic) on inequality.
# Discussion
Taken together our results show that dynamic networks promote cooperation and higher earnings, but only among prosocials. In homogeneous dynamic networks, prosocials cooperate at rates heretofore unseen in this literature. In both homogeneous and heterogeneous networks, prosocials maintain their ties longer because they cooperate more often. Greater durability of relations, in turn, leads to increased earnings. The increased earnings of prosocial actors in dynamic networks explains why these networks generate inequality, favoring prosocials.
Prior work has shown that dynamic networks promote cooperation. However, much of this work arguably conflates network dynamics with reputation processes [bib_ref] Dynamic social networks promote cooperation in experiments with humans, Rand [/bib_ref] [bib_ref] Cooperation and assortativity with dynamic partner updating, Wang [/bib_ref] [bib_ref] Quality versus quantity of social ties in experimental cooperative networks, Shirado [/bib_ref]. In these studies, when participants form new ties, they are given reputational information of potential alters. It is therefore unclear whether network dynamics, reputational processes [bib_ref] Partner choice drives the evolution of cooperation via indirect reciprocity, Roberts [/bib_ref] , or some combination of the two drives observed levels of cooperation. Here we sought to isolate the effects of pure network dynamics from reputational processes. To this end, we did not give participants any information on prospective alters' reputations. While these reputation-free networks are obviously less realistic than networks where reputations are known, they allow us to demonstrate that network dynamics alone can promote cooperation, as long as these networks contain prosocials.
Despite the fact that prosocials in heterogeneous networks could not form new ties based on reputations or with knowledge of prospective alters' dispositions, their relations became increasingly homophilous over time. The homogeneous networks of prosocials can thus be viewed as an exogenous imposition of how prosocials' networks would have evolved had we allowed the initially heterogeneous networks to play out longer. That prosocials' networks tended towards homophily and homophilous networks of prosocials cooperated at such high rates does not, however, imply that further dynamics would be unnecessary for cooperation in networks composed entirely of prosocials. Cooperation rates in homogeneous groups of prosocials were much lower in static than dynamic networks, suggesting that dynamics are also critical.
Why are dynamics needed for homogeneous networks of prosocials, despite their preference for mutual cooperation [bib_ref] The pursuit of joint outcomes and equality in outcomes: An integrative model..., Van Lange [/bib_ref] ? The answer, we suggest, centers on another fundamental issue confronted by persons in situations of interdependence like the PD: trust, or the expectation that alter will also cooperate 31 . Prior work shows that trust is the primary barrier to prosocials' cooperation [bib_ref] Social values, subjective transformations, and cooperation in social dilemmas, Simpson [/bib_ref] [bib_ref] Inducing cooperative behavior among proselfs versus prosocials: the moderating role of incentives..., Boone [/bib_ref]. Importantly, participants in our experiment could not communicate, nor did they have any information about why an alter might have not cooperated. Thus, especially in early rounds, a prosocial participant who defected out of concern that alter may not cooperate would likely trigger defection by the (prosocial) alter in the subsequent round. Indeed, looking only at the behaviors of prosocials, we find that cycles of defection, where a prosocial defects in response to alter's defection on the previous round, are 75% less likely to occur in dynamic networks (p < 0.001; SI Appendix), regardless of whether the networks were homogeneous or heterogeneous. With no recourse to severing ties, initially uncooperative acts in static networks likely triggered cycles of recrimination by other prosocials, leading to the lower cooperation observed in static homogeneous networks. Thus, it is not sufficient for populations to be high in prosociality: there must also be the possiblity of further dynamics.
Our findings shed light on the viability of prosocial dispositions and, more generally, the well-documented evidence of heterogeneous social preferences [bib_ref] Development of prosocial, individualistic, and competitive orientations: theory and preliminary evidence, Van Lange [/bib_ref] [bib_ref] Altruism and indirect reciprocity: The interaction of person and situation in prosocial..., Simpson [/bib_ref] [bib_ref] Comparing the neural basis of decision making in social dilemmas of people..., Emonds [/bib_ref]. Prosocials in homogeneous and heterogeneous networks fared substantially better than their more egoistic counterparts when they could alter their networks. This suggests that the relative viability of prosocials versus egoists in dynamic networks depends on the "stickiness" of ties, or the frequency at which actors can sever or add new relations [bib_ref] Strong ties promote the evolution of cooperation in dynamic networks, Melamed [/bib_ref] [bib_ref] Cooperation and assortativity with dynamic partner updating, Wang [/bib_ref] [bib_ref] Quality versus quantity of social ties in experimental cooperative networks, Shirado [/bib_ref]. A key question for future work is thus how this stickiness alters the balance of population level prosociality and egoism.
# Methods
The Institutional Review Board at the University of South Carolina reviewed and approved this research, and the research was conducted in accordance with their guidelines and regulations. Informed consent was obtained from all participants before data collection. We recruited a total of 1,174 participants who completed a pre-study questionnaire with the nine-item social value orientation measure (see SI Appendix). Of those surveyed, 592 were classified as prosocial, 475 were classified as egoists, and the remaining 107 could not be classified. Based on social value orientations, participants were given an access code enabling them to sign up for the experiment. The access code enabled us to sort them. Participants were not informed that their survey responses affected their eligibility. On average, participants completed the pre-study survey 26.4 days (SD = 18.5) before taking part in the experiment. The data were modeled using random intercept multilevel or mixed effects models. Cooperation was fit using the logistic link with alters nested in rounds, rounds nested in participants, and participants nested in networks. Relationship duration and earnings were fit using an identity link with instances of the outcome nested in participants, and participants nested in networks. Finally, network inequality was fit using an identity link with rounds nested in networks.
[fig] Figure 1: Marginal probability of cooperation with each alter in (A) homogeneous and (B) heterogeneous networks. Error bars refer to 95% confidence intervals around the marginal means. [/fig]
[fig] Figure 2: Marginal (A) homophily (proportion of alters that have the same Social Value Orientation as ego) and (B) number of ties (the count of ties for each participant) in heterogeneous dynamic networks. In terms of number of ties, each participant began with three ties but marginal means are shown. Error bars refer to 95% confidence intervals around the marginal means. [/fig]
[fig] Figure 3: Marginal earnings/fitness in (A) homogeneous and (B) heterogeneous networks. Error bars refer to 95% confidence intervals around the marginal means. Scientific RepoRts | 7: 357 | DOI:10.1038/s41598-017-00265-x [/fig]
[fig] Figure 4: Marginal inequality (Gini coefficients) in the heterogeneous networks. Error bars refer to 95% confidence intervals around the marginal means. [/fig]
|
Effect of Opioids on All-cause Mortality and Sustained Opioid Use in Elderly Patients with Hip Fracture: a Korea Nationwide Cohort Study
Background:The purpose of our study was to assess the use of opioids before and after hip fracture in elderly patients in order to determine the effect of opioid use on all-cause mortality, and to analyze how the history of opioid use before fracture increases the risk of sustained use following hip fracture using a Korea nationwide cohort. Methods: Our study identified hip fracture patients from the Korean National Health Insurance Service-Senior cohort. The index date was defined as 90-days after admission to the acute care hospital that fulfilled the eligibility criteria of elderly hip fracture. Patients were classified into past user, current user, and sustained user according to the use of opioid at each period based on the time of admission and index date. The opioids were classified into strong opioids and tramadol. A generalized estimating equation model with a Poisson distribution and logarithmic link function was performed to estimate the adjusted rate ratios (aRRs) and 95% confidence intervals (CIs) to assess the association between past use and sustained use. A multivariable-adjusted Cox proportional hazard model was used to investigate the effects of strong opioid and tramadol use on all-cause mortality. Results: A total of 12,927 patients were included in our study. There were 7,384 (57.12%) opioid past-users, 11,467 (88.71%) opioid current-users, and 7,172 (55.48%) sustained users. In comparison of the death risk according to current use or the defined daily dose of the opioids or past opioid use, there were no significant differences in the adjusted hazard ratio for death in all groups, compared to the current non-users (P > 0.05). Among survivors 1 year after hip fracture, opioid past-use increased the risk of opioid sustained use by 1.52-fold (aRR; 95% CI, 1.45-1.8; P < 0.001). Conclusion: Current use and past use of opioid did not increase all-cause mortality after hip fracture in elderly patients over 65 years of age. Past use of opioid before hip fracture increased risk of sustained use of opioid compared to the current opioid used without past use. contributed equally to this work.
# Introduction
In the elderly population, hip fracture is a severe injury with high morbidity and mortality.Substantial efforts are needed and medical costs are incurred in restoring function in elderly patients with hip fractures. 1,2 Expecting an increase in hip fracture patients in the future, medical and institutional efforts are being made to reduce social costs. Among these, proper pain control is important to reduce delays in ambulation and complications, and to shorten the length of hospital stay.Elderly patients with hip fracture often use various medications for underlying comorbidities and are less likely to use more drugs due to decline in organ functions, such as the liver and kidneys.Nevertheless, intravenous or oral medications, such as opioids and non-steroidal anti-inflammatory drugs (NSAIDs), are commonly used to control postoperative pain.
Opioids can cause side effects, such as dizziness, sedation, balance problems, vomiting, constipation, and respiratory depression. Long-term use can lead to physical dependence and tolerance, and worst case, to addiction.However, the use of opioids has been increasing because they are a favorable option in terms of pain control.There have been several reports on opioid use in elderly patients with hip fractures. In a Danish nationwide cohort study, the proportion of opioid users increased after hip fracture surgery, and 16.8% of preoperative non-opioid users reported using opioids even after one year. 7 A small cohort study by reported that the use of opioids was not related to mortality. A'Court et al.evaluated the relationship between opioid use and operative methods and Maiese et al.reported the cost to patients using opioids. Although these studies showed that the longterm use of opioids was not beneficial for patients, they did not analyze the effects of opioid use in the acute phase of hip fracture in elderly patients, and some of the studies were not conducted using representative big data.
Thus, the purpose of this study was to assess the use of opioids before and after hip fracture in elderly patients in order to determine the effect of opioid use on all-cause mortality, and to analyze how opioid use before fracture increased the risk of sustained use after hip fracture, using a nationwide cohort.
# Methods
## Study subjects
Incident hip fracture patients in our retrospective observational cohort study were identified from the Korean National Health Insurance Service-Senior cohort (NHIS-Senior). 9 NHIS-Senior consisted of 588,147 participants over 60 years of age (as of 2002) to represent elderly people in South Korea by simple 10% random sampling. All the individuals in the NHIS-Senior were followed until December 31, 2015, except those who were disqualified (emigration or death etc.). The NHIS has all personal information, such as medical utilization, demographics, and treatment information, for all Korean people. 9 -11 10 S721) 12,; 2) recipients of surgeries, including internal fixation (closed pinning and open reduction of a fractured extremity [femur]), hemiarthroplasty (hip), or total arthroplasty (hip); and 3) aged 65-99 years at the time of hip fracture to ensure the inclusion of osteoporotic hip fractures.To guarantee a one-year observation period, patients with hip fractures occurring less than 365 days before December 31, 2015 were excluded. Patients with their first hip fracture prior to , were also excluded to ensure at least a 3-year hip fracture-free period. The last date of follow-up was defined as the date of death or December 31, 2015, whichever preceded. To identify opioid use during the acute phase, time zero (index date) was defined as 90 days after admission to an acute care hospital to fulfill the inclusion criteria for hip fractures in the elderly. Furthermore, to prevent immortal time bias, patients who died within 90 days after hip fracture were excluded from the study.
## All-cause mortality
In the NHIS-Senior, each subject's unique de-identified number was linked to their vital statistics from the Korean National Statistical Office, including dates and causes of death.The dates of death were used to calculate survival periods.
## Categorization by type of opioid and definition of opioid exposure
The opioids used were hydromorphone, morphine, dihydrocodeine, oxycodone, hydrocodone, codeine, fentanyl, pentazocine, pethidine, buprenorphine, nalbuphine, butorphanol, tapentadol, remifentanil, sufentanil and tramadol. 7,14,The opioids were classified into strong opioids (opioids other than tramadol) and tramadol. 14 Patients who used both tramadol and strong opioids were classified as strong opioid users. Patients who had used opioids within six months before the hip fracture were defined as past users, and patients with no history of opioid use within six months before the hip fracture were defined as a past non-user. Patients who received opioids within three months after admission for hip fracture were defined as current users, and those who did not were defined as current nonusers. The cumulative dose of current tramadol and strong opioid was calculated by adding the amount of dispensed defined daily doses (DDDs).To determine the sustained use of strong opioids and tramadol, their use was investigated during the period from 3-months to 1-year after hip fracture (sustained user).
The history of medication and use of antidiabetic, antihypertensive, anti-platelet agent, NSAIDs, steroids, COX-2 inhibitors, antidepressants, and benzodiazepines within 6-months before hip fracture and within 3-months after admission for hip fracture were investigated.
# Statistical analysis
Demographics were identified at the time of admission. Survival time was defined in days as the period from the index date (at 90-days after admission) to the date of December 31, 2015 or death, whichever preceded. Patients who died during the 90-day landmark period were excluded. A multivariable-adjusted Cox proportional hazard model was used to investigate the effects of tramadol and strong opioid use on all-cause mortality. The effect sizes are presented as hazard ratios (HRs) and 95% confidence intervals (CIs). A generalized estimating equation model with a Poisson distribution and logarithmic link function was used to estimate the adjusted rate ratios (aRRs) and 95% CIs to assess the association between past use and sustained use. The potential confounders included age group, gender, household income level, residential area, Charlson Comorbidity Score (CCS), type of fracture, surgery and anesthesia, calendar year of the hip fracture incidence, presence of transfusions, medication history, and recent medication use, which included strong opioids 3/11 https://jkms.org https://doi.org/10.3346/jkms.2021.36.e127
## Opioids in elderly hip fracture
and tramadol. Quan's ICD-10 coding algorithm of the CCS diagnostic codes during the 3 years before admission for hip fracture was used to assess each subject's comorbidities.The presence of CCS disease-constituting categories was calculated by the presence of at least 2 outpatient visits or 1 admission for primary or first secondary diagnosis. Statistical analyses were conducted using SAS Enterprise Guide version 7.1 (SAS Institute, Cary, NC, USA). Statistical significance was set at P < 0.05.
# Ethics statement
The study design and protocol were approved by the Institutional Review Board of our hospital (EMC-IRB No 2018-01-010-001). Written informed consent was waived for all patients involved in our study.
# Results
Between January 1st, 2002, and December 31st, 2015, 19,915 patients with hip fractures were identified. Among these patients, 1,478 patients did not undergo surgery and 3,571 patients were excluded for not meeting the eligible criteria. In addition, 921 patients with a history of neoplasms and 1,018 patients who died within 90-days of hip fracture were excluded. Ultimately, 12,927 patients were included in our study . The patient demographics and medication history and recent medication use are presented in . There were no significant differences in the cumulative mortality rates between the two groups (P < 0.05). opioid and tramadol current users increased in comparison to the proportion of past users in both categories, respectively. However, there was a greater increase in the number of tramadol current users than in strong opioid current users. The proportion of sustained users decreased to a level similar to that of past users, when compared to the proportion of current users . The number of new users of tramadol and strong opioids among past non-users also increased after hip fracture. Moreover, the proportion of sustained users among new opioid users in the past non-user group decreased after one year. On death risk comparison according to current use and the DDD of strong opioids and tramadol in each patient group according to past opioid use, there were no significant differences in the adjusted HR compared to the current non-users in all groups (P > 0.05) ( Among those who survived up to one year after hip fracture (1,376 patients died before 1-year after hip fracture), opioid past-use increased the risk of opioid sustained use by 1.52-fold (aRR; 95% CI, 1.45-1.8; P < 0.001). Tramadol and strong opioid past-use in current users increased the risk of a sustained use of tramadol and strong opioid by 1.49-fold (aRR; 95% CI, 1.43-1.56; P < 0.001) and 1.57-fold (aRR; 95% CI, 1.4-1.76; P < 0.001), respectively, compared to past non-users .
# Discussion
The main findings of this study are as follows. First, the current and past use of opioids did not increase all-cause mortality after hip fractures in elderly patients. Second, the opioid prescription rate increased rapidly within three months after hip fracture compared to before the hip fracture, but decreased three months after hip fracture. Third, current users with a history of opioid use had a higher risk of sustained opioid use compared to those who did not. In 1995, the American Pain Society reported pain as a fifth vital sign that should be treated appropriately.This change in the concept of drug prescriptions for pain control increased the prescription of opioids, resulting in increased patient satisfaction. Since then, opioids have become an essential component of multimodal postoperative analgesia and are still widely used.However, this trend has led to opioid abuse for chronic musculoskeletal pain in the United States and Europe, and has emerged as a social problem. 20, reported that the use of opioids for non-cancer pain in a Danish population was highest in those aged 65 years and older. Several studies have shown that the side effects of opioids, such as cognitive impairment, dizziness, addiction, mortality, increased fall and fracture risk, are more likely to occur in elderly patients. 22,Paradoxically, insufficient pain control can lead to delirium, day-night inversion, and depression. 2
Opioid-related deaths are among the most frequent types of drug-related deaths.Summers et al.reported that hip fracture patients with opioid use disorders had higher mortality rates than hip fracture patients with non-opioid use disorders. Patients with opioid use disorder had more comorbidities than those who did not. Therefore, they should be distinguished from patients with a history of opioid use. Lindestrand et al.investigated the use of opioids in patients with hip fractures and the potential relationship between the perioperative prescription of opioids, mortality, and chronic opioid use. In their study, the 30-day mortality rate was 10% and 1-year mortality rate was 27%. They found that mortality was associated with old age and increased comorbidities, but was not associated with opioid use. Therefore, they suggested that there was no general reason to refrain from prescribing opioids to hip fracture patients based on a fear of increased mortality. Our results are consistent with these findings. Tramadol has a lower analgesic potency than strong opioids, but is relatively safer due to a lower risk of respiratory depression and addiction.Therefore, we expected that all-cause mortality would be higher in the group using potent opioids at follow-up in both groups. However, neither tramadol nor strong opioids increased all-cause mortality. In addition, we found that opioid use before and after hip fracture did not contribute to increased mortality. Our study also showed that there was no increase in mortality even in groups taking high opioid doses after hip fracture, when compared to the non-user group. In elderly patients, the combination of several drugs is an important issue, and there are reports that the combination of central nervous system active drugs, including benzodiazepines and tricyclic antidepressants, with opioids can increase mortality. 27,28 However, our study found that opioids, after adjusting for several other drugs, including benzodiazepines and tricyclic antidepressants, did not significantly affect postoperative mortality.
Most recently, a large epidemiological study on opioid use after hip fracture surgery was published in Denmark. The authors performed a cohort study that included all patients (65 years old and over) who underwent hip fracture surgery (from 2005 to 2015), as selected from the Danish Multidisciplinary Hip Fracture Database. 7 In their study, similar to the results of our study, the rate of opioid users increased rapidly after hip fracture and then decreased. They also found that the proportion of opioid users increased after hip fracture surgery and increased by 1.4% in the fourth quarter compared to before hip fracture. Of the opioid nonusers before surgery, 16.8% were opioid users in the fourth quarter after surgery. They suggested that opioid use at 1-year after surgery for hip fracture was common, both in patients who were opioid users and non-users before surgery. However, opioids are essential for postoperative pain control. In addition, depending on the degree of fracture reduction or the result of surgery, chronic pain or complications may persist, and pain control may be Opioids in Elderly Hip Fracture necessary. Therefore, it is necessary to detect and be aware of the risk of future abuse among patients who currently need opioids.
[formula] 8 [/formula]
Simoni et al.reported that 8.3% of opioid non-users and 52.3% of opioid users before surgery used opioids one year after surgery. Morris and Mir 29 also reported that pre-injury opioid users among orthopedic trauma patients had a high risk of postoperative opioid abuse. In addition, the use of preoperative opioids among orthopedic patients has been reported to cause poor clinical outcomes and increase the risk of reoperation. 30,We also found that among the current opioid users within three months after fracture, patients with a history of opioid use within six months prior to the fracture had a higher risk of sustained opioid use. This finding is in agreement with the results of a study that reported that preoperative opioid experience could increase the likelihood of opioid abuse. 32 This may be due to dependence of past opioid use or opioid-induced hyperalgesia, making it difficult to discontinue opioids.Thus, before opioids are used after hip fracture surgery, a history of opioid use before the fracture should be carefully noted. Furthermore, for past opioid users, care should be taken not to prescribe opioids for a long period of time. In addition, it is necessary to follow-up to assess dependence or addiction in patients using opioids after surgery.
This study has several limitations. First, there was no analysis of functional or pain scores for hip fracture treatment. Therefore, there was no analysis of the effect of opioids on functional aspects or pain control. Second, since this study was solely based on a South Korean database, it may have had potential biases, such as regional and cultural biases. Spain and Sweden are both European countries, but the rate of opioid use before hip fracture was different between the two countries. As such, the guidelines for opioid use and medical systems are different in each country.However, this study compared the relative risk of mortality and the sustained use of opioids between subgroups. Third, our study did not define addiction. Drug addiction is not simply defined by dosage, but various factors such as dependence must be considered. Fourth, the specific cause of death could not be analyzed because of inherent limitations in the claims database.
In conclusion, the current use and past use of opioids did not increase all-cause mortality after hip fracture in elderly patients over 65 years of age. In current opioid users, past use of opioids before hip fracture increased the risk of sustained use of opioids compared to those without. |
PCSK9 inhibitors: A new era of lipid lowering therapy
Hyperlipidemia is a well-established risk factor for developing cardiovascular disease (CVD). The recent American College of Cardiology and American Heart Association guidelines on lipid management emphasize treatment of individuals at increased risk for developing CVD events with 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) at doses proven to reduce CVD events. However, there are limited options for patients who are either intolerant to statin therapy, develop CVD despite being on maximally tolerated statin therapy, or have severe hypercholesterolemia. Recently the Food and Drug Administration approved two novel medications for low-density lipoprotein (LDL)-cholesterol reduction: Evolocumab and Alirocumab. These agents target and inactivate proprotein convertase subtilsinkexin type 9 (PCSK9), a hepatic protease that attaches and internalizes LDL receptors into lysosomes hence promoting their destruction. By preventing LDL receptor destruction, LDL-C levels can be lowered 50%-60% above that achieved by statin therapy alone. This review explores PCSK-9 biology and the mechanisms available to alter it; clinical trials targeting PCSK9 activity, and the current state of clinically available inhibitors of PCSK9.
# Introduction
Hyperlipidemia is a well-established risk factor for developing cardiovascular disease (CVD). Multiple double blind placebo controlled trials have shown that treatment with HMG CoA Reductase inhibitors (statins) lowers low-density lipoprotein (LDL)-C levels and reduces CVD events in individuals with CVD or those at high risk for developing it [bib_ref] Statins for the primary prevention of cardiovascular disease, Taylor [/bib_ref]. However, CVD events continue to occur in some patients on statins, despite receiving maximal tolerated therapy. Other patients develop side effects from statins that limit their use. Hence, newer modalities of treatment to lower LDL-C are needed in clinical practice. Recently the Food and Drug Administration (FDA) approved two medications which target a novel pathway to reduce LDL-C. They are monoclonal antibodies that inactivate proprotein convertase subtilsin-kexin type 9 (PCSK9). This review will explore the biology of PCSK 9, clinical trials targeting PCSK9 activity, and the current state of clinically available inhibitors of PCSK9.
## Ldl-cholesterol metabolism
LDL-C has been the target of therapy for improving outcomes in patients at high risk for developing CVD and has been considered a surrogate endpoint for clinical events by the FDA. Reviewing LDL cholesterol metabolism is therefore important in understanding therapeutic approaches to treat hyperlipidemia.
The lipid cycle begins with the release of immature very low-density lipoprotein (VLDL) or nascent VLDL from the liver. Nascent VLDL contains apolipoprotein-B100 (apoB-100), apolipoprotein E (apoE), apolipoprotein C1 (apoC1), cholesteryl esters, cholesterol, and triglycerides. While circulating in blood, high-density lipoprotein (HDL) donates apolipoprotein C-Ⅱ (apoC-Ⅱ) to nascent VLDL that leads to its maturation. Mature VLDL interacts with lipoprotein lipase (LPL) in the capillary beds of adipose tissues, cardiac muscle and skeletal muscle cells, which leads to extraction of triglycerides from VLDL for storage or energy production in these tissues. VLDL combines with HDL again and an interchange occurs where apoC-Ⅱ is transferred back to HDL along with phospholipids and triglycerides in exchange for cholesteryl esters via cholesterylester-transfer protein (CETP). This exchange and removal of triglycerides leads to conversion of VLDL to intermediate-density lipoprotein (IDL) [bib_ref] Very-low-density lipoprotein assembly and secretion, Shelness [/bib_ref]. Half of IDLs are recognized and endocytosed by liver cells due to apoB-100 and apoE. The remaining IDL lose apoE, and with an increased concentration of cholesterol compared to triglyceride, transform into low-density lipoproteins (LDL). LDL particles thus formed contain apoB-100, which acts as a ligand for binding to LDL receptors (LDLR). Once LDL binds to LDLR, LDL/LDLR complex is internalized by endocytosis into clathrin coated vesicles. In the cytosol, LDL and LDLR separate with recycling of LDLR to the cell surface. LDLR recycling is a continuous process and each receptor recycles up to 150 times after which they are endocytosed and metabolized . Statins act by inhibiting 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, which is involved in intracellular production of cholesterol. This lowers the levels of intracellular cholesterol leading to increased expression of LDLR on cell surfaces causing a reduction in serum LDLcholesterol [bib_ref] Statins: mechanism of action and effects, Stancu [/bib_ref]. Seidah and colleagues discovered that proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates LDLR degradation and could potentially be a target for modulating LDLR expression and consequently LDL-C levels [bib_ref] The secretory proprotein convertase neural apoptosis-regulated convertase 1 (NARC-1): liver regeneration and..., Seidah [/bib_ref] [bib_ref] Mutations in PCSK9 cause autosomal dominant hypercholesterolemia, Abifadel [/bib_ref]. PCSK9 is a hepatic protease that attaches to and internalizes LDLR into lysosomes hence promoting their destruction [bib_ref] The PCSK9 decade, Lambert [/bib_ref]. Clinical studies have shown that PCSK9 gain of function mutation is associated with familial hypercholesterolemia and premature CVD [bib_ref] Proprotein convertase subtilisin/kexin type 9 (PCSK9): from structure-function relation to therapeutic inhibition, Tibolla [/bib_ref] [bib_ref] The influence of PCSK9 polymorphisms on serum low-density lipoprotein cholesterol and risk..., Davignon [/bib_ref]. Conversely, individuals with loss of function mutations in PCSK9 have been observed to have lower lifetime levels of LDL-C and lower prevalence of CVD [bib_ref] Low LDL cholesterol in individuals of African descent resulting from frequent nonsense..., Cohen [/bib_ref] [bib_ref] Sequence variations in PCSK9, low LDL, and protection against coronary heart disease, Cohen [/bib_ref].
Since the discovery of PCSK9, results from preclinical mice studies demonstrated that sterol regulatory element binding protein-2 (SREBP-2) plays a key role in regulating cholesterol metabolism. Low level of intracellular cholesterol activates SREBP-2 and leads to LDLR gene expression. This increases LDLR concentration thus enhancing LDL clearance from circulation [bib_ref] Mutations in PCSK9 cause autosomal dominant hypercholesterolemia, Abifadel [/bib_ref] [bib_ref] Gene inactivation of proprotein convertase subtilisin/kexin type 9 reduces atherosclerosis in mice, Denis [/bib_ref]. At the same time SREBP-2 also induces the expression of PCSK9, which promotes LDLR degradation. Thus, the coordinated interplay of SREBP-2 induced transcription of both LDLR and PCSK9 regulates circulating LDL levels [bib_ref] Novel putative SREBP and LXR target genes identified by microarray analysis in..., Maxwell [/bib_ref] [bib_ref] Combined analysis of oligonucleotide microarray data from transgenic and knockout mice identifies..., Horton [/bib_ref]. These discoveries resulted in the exploration and development of therapeutic agents to lower LDL levels by targeting PCSK9 activity.
## Functional mechanics of pcsk9
Hepatocytes are the predominant site for PCSK9 production, with other sites being intestines and kidneys [bib_ref] Decreased plasma cholesterol and hypersensitivity to statins in mice lacking Pcsk9, Rashid [/bib_ref] [bib_ref] Proprotein convertase subtilisin/kexin type 9 (PCSK9): hepatocyte-specific low-density lipoprotein receptor degradation and..., Zaid [/bib_ref]. PCSK9 reduces the number of LDLR in hepatocytes by promoting their metabolism and subsequent degradation [bib_ref] Gene inactivation of proprotein convertase subtilisin/kexin type 9 reduces atherosclerosis in mice, Denis [/bib_ref]. PCSK9 has been shown to act both intracellularly (playing a role as a chaperone) as well as a secreted factor promoting LDLR internalization from the hepatocellular surface. Under normal circumstances, the LDL/LDLR complex is endocytosed by endosomes. The acidic pH of the endosome reduces the affinity of LDL for LDLR with rearrangement of the LDLR's extracellular domain into a hairpin structure, aiding in its recycling back to plasma membrane. PCSK9 binding inhibits this change and locks the LDLR in an open conformation which prevents its recycling. The LDLR is then routed to lysosomes for degradation [fig_ref] Figure 1: Mechanism and role of PCK9 in low-density lipoprotein-cholesterol metabolism [/fig_ref] [bib_ref] Mechanistic implications for LDL receptor degradation from the PCSK9/LDLR structure at neutral..., Lo Surdo [/bib_ref] [bib_ref] A two-step binding model of PCSK9 interaction with the low density lipoprotein..., Yamamoto [/bib_ref]. The secreted form of PCSK9 circulates in the bloodstream and can be inactivated by cleavage from proprotein convertase. At a molecular level, the secretion of prodomain and catalytically inactive PCSK9 promotes regular degradation of LDLR implying that PCSK9 acts as a chaperone protein rather than an active catalytic enzyme [bib_ref] Secreted PCSK9 promotes LDL receptor degradation independently of proteolytic activity, Li [/bib_ref] [bib_ref] Catalytic activity is not required for secreted PCSK9 to reduce low density..., Mcnutt [/bib_ref]. As described above, hepatic expression of PCSK9 and LDLR are closely regulated by SREBP-2 and intracellular levels of cholesterol [bib_ref] Strong induction of PCSK9 gene expression through HNF1alpha and SREBP2: mechanism for..., Dong [/bib_ref] [bib_ref] Hepatocyte nuclear factor 1alpha plays a critical role in PCSK9 gene transcription..., Li [/bib_ref]. Lipid lowering therapy with statins [bib_ref] Atorvastatin increases human serum levels of proprotein convertase subtilisin/kexin type 9, Careskey [/bib_ref] [bib_ref] proprotein convertase subtilisin/ kexin type 9 concentrations, and LDL cholesterol response: the..., Awan [/bib_ref] [bib_ref] High-dose atorvastatin causes a rapid sustained increase in human serum PCSK9 and..., Welder [/bib_ref] , ezetimibe [bib_ref] Statins and ezetimibe modulate plasma proprotein convertase subtilisin kexin-9 (PCSK9) levels, Davignon [/bib_ref] and bile acid binding resins [bib_ref] Bile acids and lipoprotein metabolism: effects of cholestyramine and chenodeoxycholic acid on..., Nilsson [/bib_ref] cause induction of SREBP-2 and hence co-induces both PCSK9 and LDLR. The slight increase in PCSK9 activity seen with statins does not negate their therapeutic effectiveness.
## Other functions and locations of
## Pcsk9
Apart from hepatocytes, PCSK9 is also expressed in intestine, central nervous system, and mesenchymal cells of the kidney. In vitro studies on human intestinal epithelium have reported recombinant PCSK9 to enhance cholesterol uptake in the human intestinal epithelial cells (Caco-2/15 cell line) via the up regulation of the protein expression of NPC1L1 and CD36 (involved in cholesterol absorption in intestinal cells) along with an increased expression of cholesterol transporters [bib_ref] Niemann-pick C1-like 1 (NPC1L1) protein in intestinal and hepatic cholesterol transport, Jia [/bib_ref] and reduced cholesterol synthesis (by reducing HMG-CoA reductase activity) [bib_ref] PCSK9 plays a significant role in cholesterol homeostasis and lipid transport in..., Levy [/bib_ref]. PCSK9 has been shown to have a role in the metabolism of triglycerides and their accumulation in visceral adipose tissue [bib_ref] Circulating proprotein convertase subtilisin/kexin 9 (PCSK9) regulates VLDLR protein and triglyceride accumulation..., Roubtsova [/bib_ref]. It also promotes chylomicron secretion and helps regulate enterocyte cholesterol [bib_ref] Anti-PCSK9 antibody effectively lowers cholesterol in patients with statin intolerance: the GAUSS-2..., Stroes [/bib_ref] February 26, 2017|Volume 9|Issue 2| WJC|www.wjgnet.com balance [bib_ref] PCSK9 plays a significant role in cholesterol homeostasis and lipid transport in..., Levy [/bib_ref]. Studies have evaluated PCSK9 and their association with increased susceptibility to hepatitis C viral infection. Labonté et al [bib_ref] PCSK9 impedes hepatitis C virus infection in vitro and modulates liver CD81..., Labonté [/bib_ref] demonstrated a reduced expression of CD81 (CD81 is a co-receptor for Hepatitis C virus infection) by PCSK9 leading to protection against infection by hepatitis C. Therefore, PCSK9 inhibitors (Alirocumab) could increase CD81 expression resulting in greater infectivity. However, in vitro and in vivo studies in mice showed that PCSK9 did not reduce CD81 expression and had no effect on HCV infectivity. Hence, the literature remains inconclusive about this potential effect of PCSK9 inhibition [bib_ref] PCSK9 impedes hepatitis C virus infection in vitro and modulates liver CD81..., Labonté [/bib_ref]. Mbikay et al [bib_ref] PCSK9-deficient mice exhibit impaired glucose tolerance and pancreatic islet abnormalities, Mbikay [/bib_ref] demonstrated that PCSK9 inhibition in mouse pancreatic islet β cells led to hypoinsulinemia, hyperglycemia and glucoseintolerance. Additionally the islet cells exhibited signs of malformation, apoptosis and inflammation. Current clinical data has failed to show this as a complication with PCSK9 inhibition.
## Pcsk9 inhibition strategies
Preclinical studies demonstrate that statin-induced LDL reduction occurs through increased LDLR expression on hepatocytes along with increased LDL turnover, which leads to its enhanced clearance from the circulation. However, statins also induce PCSK9 expression, which dampens the effective LDL clearing by promoting LDLR degradation [bib_ref] Strong induction of PCSK9 gene expression through HNF1alpha and SREBP2: mechanism for..., Dong [/bib_ref]. Since PCSK9 is expressed both intracellularly and in the circulation, multiple potential targets exist for its inhibition. Various modalities to inhibit PCSK9 have been studied including: (1) inhibition of production by gene silencing through antisense oligonucleotides [bib_ref] Antisense inhibition of proprotein convertase subtilisin/kexin type 9 reduces serum LDL in..., Graham [/bib_ref] or small interfering RNA [bib_ref] Therapeutic RNAi targeting PCSK9 acutely lowers plasma cholesterol in rodents and LDL..., Frank-Kamenetsky [/bib_ref] ;
(2) prevention of PCSK9 binding to LDLR using monoclonal antibodies [bib_ref] Antibody-mediated disruption of the interaction between PCSK9 and the low-density lipoprotein receptor, Duff [/bib_ref] , epidermal growth factor-like repeat A (EGF-A), mimetic peptides [bib_ref] PCSK9 binds to multiple receptors and can be functionally inhibited by an..., Shan [/bib_ref] or adnectins; and (3) inhibition of PCSK autocatalytic sites. to statin therapy in patients with high atherosclerotic cardiovascular risk with a follow up period of up to 12 wk. SPIRE-1 (NCT01975376) and 2 (NCT01975389) have a follow up period of up to 5 years collecting data on safety and efficacy of this drug [bib_ref] Lipid lowering with PCSK9 inhibitors, Dadu [/bib_ref]. Recently, the preliminary results of a study of Bococizumab delivery using an autoinjector device (SPIRE-AI) reported successfully meeting co-primary endpoints of percent change from baseline in fasting LDL-C at week 12 and the delivery system success rate, defined as the percent of patients whose attempts to operate the pre-filled pen. SPIRE-AI is a 12-wk, doubleblind, placebo-controlled, randomized, parallel-group, multicenter, phase Ⅲ clinical trial in 299 patients with hyperlipidemia or mixed dyslipidemia receiving statin therapy and whose LDL-C ≥ 70 mg/dL and assessed the efficacy, safety, tolerability and subcutaneous administration of Bococizumab 150 mg and 75 mg with a prefilled pen. Along similar lines, adnectins (also known as monobody) and small peptide inhibitors have been investigated for LDL reduction in phase Ⅰ clinical trials. The results in healthy patients and those with hypercholesterolemia demonstrated a maximal dose related reduction of LDL-C by up to 48% [bib_ref] LDL cholesterol reduction with bms-962476, an adnectin inhibitor of PCSK9: results of..., Stein [/bib_ref]. The advantage of developing adnectins is that they are smaller than mAb, making them cheaper and easier to produce. Their pharmacokinetics have been shown to be favorable with a rapid onset of action in preclinical models, further trials are awaited to see the development of this agent [bib_ref] Pharmacologic profile of the Adnectin BMS-962476, a small protein biologic alternative to..., Mitchell [/bib_ref].
## Inhibition of autocatalytic site
This mechanism as a therapeutic target was first proposed after discovering a loss of function mutation in the autocatalytic cleavage site of PCSK9 [bib_ref] Low density lipoprotein binds to proprotein convertase subtilisin/kexin type-9 (PCSK9) in human..., Kosenko [/bib_ref] [bib_ref] Abstract 15853: Identification of Novel Sites Required for PCSK9 Autocatalytic Cleavage, Huang [/bib_ref]. This approach is still under preclinical investigational phase.
## Fda approval status of pcsk9
## Inhibitors
The FDA approved Alirocumab (Praluent) in July 2015 for adult patients with heterozygous familial hypercholesterolemia or in patients with clinically significant atherosclerotic CVD requiring additional LDL lowering after being on diet control and maximally tolerated statin therapy. Evolocumab (Repatha) was also approved in August, 2015 for use in adult patients with heterozygous familial hypercholesterolemia, homozygous familial hypercholesterolemia, or clinical atherosclerotic CVD requiring additional lowering of LDL cholesterol after being on a controlled diet and maximally-tolerated statin therapy.
## Alirocumab (status: fda approved)
Alirocumab has been studied in three phase Ⅰ trials. In two of these trials, healthy volunteers were administered Alirocumab intravenously (n = 40) or subcutaneously (n = 32) which reduced LDL-C in a dose-dependent fashion with a reduction of up to 65% at maximal doses [bib_ref] Effect of a monoclonal antibody to PCSK9 on LDL cholesterol, Stein [/bib_ref]. The
## Gene silencing via antisense oligonucleotides or small interfering rna
This approach targets intracellular PCSK9 activity by utilizing antisense oligonucleotide to reduce intracellular expression of PCSK9. Preclinical trials on hyperlipidemic mice evaluating two such compounds were promising with a reduction in LDL by 38% at six weeks of therapy while doubling LDLR expression in the liver [bib_ref] Antibody-mediated disruption of the interaction between PCSK9 and the low-density lipoprotein receptor, Duff [/bib_ref]. However, the phase Ⅰ trials evaluating two of these agents were terminated prematurely and further development of the drug (BMS-84442) was not continued (NCT01082562). Two additional drugs: SPC5001 and SPC4061 showed successful reduction in LDL by 50% during preclinical testing in primates [bib_ref] PCSK9 LNA antisense oligonucleotides induce sustained reduction of LDL cholesterol in nonhuman..., Lindholm [/bib_ref]. However, the first phase Ⅰ trial in healthy human subjects and individuals with familial hypercholesterolemia were terminated early (NCT01 350960) [bib_ref] Anti-PCSK9 therapies for the treatment of hypercholesterolemia, Hooper [/bib_ref] [bib_ref] A locked nucleic acid antisense oligonucleotide (LNA) silences PCSK9 and enhances LDLR..., Gupta [/bib_ref]. SPC5001 was seen to cause mild to moderate injection site reactions and renal tubular toxicity [bib_ref] Antisense-mediated reduction of proprotein convertase subtilisin/kexin type 9 (PCSK9): a first-in-human randomized,..., Van Poelgeest [/bib_ref]. Further development of SPC4061 was discontinued for undisclosed reasons.
Similarly, small interfering RNA (siRNA) administration has been shown to significantly reduce plasma PCSK9 and LDL levels in cynomolgus monkeys [bib_ref] Therapeutic RNAi targeting PCSK9 acutely lowers plasma cholesterol in rodents and LDL..., Frank-Kamenetsky [/bib_ref] [bib_ref] Antibody-mediated disruption of the interaction between PCSK9 and the low-density lipoprotein receptor, Duff [/bib_ref] [bib_ref] Antisense-mediated reduction of proprotein convertase subtilisin/kexin type 9 (PCSK9): a first-in-human randomized,..., Van Poelgeest [/bib_ref]. ALN-PCS is a siRNA, which was tested by delivery through secondgeneration lipid nanoparticles. In a study by Fitzgerald et al, ALN-PCS demonstrated a dose dependent reduction in PCSK9 and LDL levels with a reduction of up to 70% in PCSK9 levels and 40% in LDL levels at doses of 0.4 mg/kg. This was the first study to demonstrate intracellular PCSK9 inhibition translated into reduction of circulating LDL levels.
## Monoclonal antibodies
Utilization of monoclonal antibodies has been the most effective approach thus far in inhibiting PCSK9 and reducing LDL levels. Currently, at least six monoclonal antibodies (mAb) have been or are being developed and tested: Alirocumab (formerly called SAR236553/ REGN727), Evolocumab (formerly called AMG145), RG7 652 [bib_ref] Modeling and Simulation to Support Phase 2 Dose Selection for RG7652, a..., Budha [/bib_ref] , LGT209 (NCT01979601, NCT01859455), 1B20 [bib_ref] An anti-PCSK9 antibody reduces LDL-cholesterol on top of a statin and suppresses..., Zhang [/bib_ref] and Bococizumab (formerly called RN316/PF-049 50615). Alirocumab and Evolocumab have recently been approved by the FDA. The major clinical studies leading up to their approval are outlined later in this paper.
Bococizumab is a unique mAb, which utilizes pH sensitive binding to PCSK9 and was developed for a longer serum half-life and duration of action on LDL reduction [bib_ref] Increasing serum half-life and extending cholesterol lowering in vivo by engineering antibody..., Chaparro-Riggers [/bib_ref].
In phase Ⅰ studies, single intravenous or subcutaneous dosages significantly reduced LDL in patients with hypercholesterolemia, both with and without concomitant atorvastatin therapy [bib_ref] Effects of 12 weeks of treatment with RN316 (PF-04950615), a humanized IgG2_a..., Gumbiner [/bib_ref]. In a phase Ⅱ trial by Gumbiner et al [bib_ref] Effects of 12 weeks of treatment with RN316 (PF-04950615), a humanized IgG2_a..., Gumbiner [/bib_ref] , patients on statin therapy not at target LDL-C were enrolled and observed a 60% reduction in LDL-C after 12 wk of therapy. Five phase Ⅲ trials are ongoing for Bococizumab including SPIRE-HF (evaluating the efficacy of this agent in heterozygous familial hypercholesterolemia NCT01968980); SPIRE-HR (NCT01968954) and SPIRE-LDL (NCT01968967) trials are comparing Bococizumab dose or switching to rosuvastatin. Atorvastatin at 20 mg and 40 mg/d regimens reduced LDL by 44% and 54% with addition of Alirocumab respectively vs 21% and 23% with addition of ezetimibe respectively vs 5% and 5% with doubling atorvastatin dose respectively compared to 21% with switching to rosuvastatin 40 mg/d. In the OPTIONS Ⅱ trial, rosuvastatin was studied using a similar protocol and showed that the addition of Alirocumab had the most significant reduction in LDL-C after 24 wk of therapy as opposed to addition of ezetimibe or doubling the dose of rosuvastatin [bib_ref] Efficacy and safety of alirocumab as add-on therapy in high-cardiovascular-risk patients with..., Robinson [/bib_ref]. Another study evaluating the efficacy of Alirocumab as monotherapy (MONO study) [bib_ref] A 24-week study of alirocumab as monotherapy versus ezetimibe: the first phase..., Roth [/bib_ref] compared to ezetimibe in patients with hypercholesterolemia and moderate cardiovascular risk to monotherapy with Alirocumab and found that Alirocumab reduced LDL-C 47% vs 16% by ezetimibe after 24 wk of therapy (P < 0.0001).
CHOICE Ⅱ study evaluated Alirocumab in patients intolerant to statin therapy. Two hundred and thirty one patients with a history of statin intolerance were shown to have a 56% reduction in LDL with Alirocumab (vs placebo; P < 0.001). Another study evaluating Alirocumab in patients intolerant to statin therapy is the ODYSSEY ALTERNATIVE trial. Three hundred and fourteen patients completed this randomized controlled trial, which compared Alirocumab 75 mg every 2 wk (n = 126) to ezetimibe 10 mg/d (n = 125) and atorvastatin 20 mg/d (n = 63) for 24 wk. At 24 wk, the data showed a 45% reduction in LDL with Alirocumab as opposed to 15% reduction in LDL with ezetimibe. This trial demonstrated fewer skeletal muscle adverse events in the Alirocumab group as compared to atorvastatin arm [32.5% vs 46% respectively, HR = 0.61 (0.38-0.99; P = 0.042)], with no significant difference when compared to the ezetimibe group (41%) [HR 0.71 (0.47 to 1.06; P = 0.09)] [bib_ref] Efficacy and safety of alirocumab vs ezetimibe in statin-intolerant patients, with a..., Moriarty [/bib_ref]. Alirocumab has also been shown to be effective in lowering LDL-C in patients with familial hypercholesterolemia. The FH Ⅰ and FH Ⅱ studies evaluated a total of 735 patients (n = 486 and 249 respectively) with heterozygous familial hypercholesterolemia inadequately controlled on lipid lowering therapy and found Alirocumab to reduce LDL levels 48.8% (vs a 9.1% increase in placebo: FH Ⅰ study) and 48.7% (vs 2.8% increase in LDL with placebo: FH Ⅱ study) from baseline [bib_ref] 78 week results with alirocumab treatment in 735 patients with heterozygous familial..., Kastelein [/bib_ref].
ODYSSEY HIGH FH trial reported 105 patients with familial hypercholesterolemia on maximally tolerated statin therapy and LDL > 160 demonstrating a 46% reduction of LDL (vs 7% with placebo) at 24 wk (P < 0.001). OLE trial (NCT01954394) is currently ongoing with results anticipated by June 2017. This trial is recruiting patients with heterozygous familial hypercholesterolemia who have completed one of the other studies and evaluating for safety parameters including adverse events, laboratory data and vital signs.
ODYSSEY LONG TERM trial was recently published and is a 78-wk follow-up of 2341 patients with hypercholesterolemia and LDL > 70 mg/dL on maximally tolerated statins. The patients receiving 150 mg Alirocumab every 2 wk were shown to have a 62% reduction third trial evaluated patients with non-familial hypercholesterolemia on atorvastatin and LDL > 100 mg/dL or with LDL > 130 mg/dL being managed by diet alone. Alirocumab reduced LDL-C up to 65% in patients on statins and up to 60% in patients being managed on diet alone. It was observed that Alirocumab remained effective for a longer period of time in patients not on statins [bib_ref] Effect of a monoclonal antibody to PCSK9 on LDL cholesterol, Stein [/bib_ref]. Three phase Ⅱ trials [bib_ref] Safety and efficacy of a monoclonal antibody to proprotein convertase subtilisin/kexin type..., Mckenney [/bib_ref] [bib_ref] Atorvastatin with or without an antibody to PCSK9 in primary hypercholesterolemia, Roth [/bib_ref] [bib_ref] Effect of a monoclonal antibody to PCSK9, REGN727/ SAR236553, to reduce low-density..., Stein [/bib_ref] evaluated the efficacy of Alirocumab in patients with familial hypercholesterolemia. Stein et al [bib_ref] Effect of a monoclonal antibody to PCSK9, REGN727/ SAR236553, to reduce low-density..., Stein [/bib_ref] evaluated 77 patients on statin therapy
with LDL-C greater than 100 mg/dL and found that Alirocumab reduced LDL-C in a dose-dependent manner by up to 43% with a maximum dosage of 300 mg every 4 wk. However, the reduction was even greater (up to 70%) at a dosage regimen of 150 mg every 2 wk. Additionally, these patients had a significant reduction in apoB levels and non-high density lipoprotein cholesterol and also had increases in HDL. McKenney et al [bib_ref] Safety and efficacy of a monoclonal antibody to proprotein convertase subtilisin/kexin type..., Mckenney [/bib_ref] , in their study of 183 patients confirmed a dose dependent reduction of LDL-C with the most efficacious regimen being 150 mg every 2 wk (with LDL reduction up to 70%). Interestingly, different doses of atorvastatin did not make a significant difference in LDL-C reduction. These results were further corroborated by Roth et al [bib_ref] Atorvastatin with or without an antibody to PCSK9 in primary hypercholesterolemia, Roth [/bib_ref] in their study of 92 patients with LDL-C > 100 mg/dL. They showed that there was significant and comparable LDL reduction irrespective of the dosage of atorvastatin (10 mg vs 80 mg) when added to Alirocumab 150 mg every 2 wk [bib_ref] Atorvastatin with or without an antibody to PCSK9 in primary hypercholesterolemia, Roth [/bib_ref]. The phase Ⅲ randomized, double-blinded ODYSSEY trials were designed to evaluate Alirocumab for longterm safety, efficacy and adverse events CHOICE Ⅰ study evaluated Alirocumab vs placebo in 803 patients with poorly controlled hypercholesterolemia. Alirocumab reduced LDL by 52% in statin-naïve patients and by 59% in patients on maximally tolerated statins as compared to placebo (P < 0.001). Similarly, COMBO Ⅰ and Ⅱ trials evaluated 316 patients and 720 patients respectively with LDL > 70 mg/dL and high cardiovascular risk on maximally tolerated statin therapy. COMBO Ⅰ showed Alirocumab reduced LDL-C up to 50% (vs 2% with placebo) after 24 wk of treatment. COMBO Ⅱ compared ezetimibe to Alirocumab in patients on background statin therapy and found a 50% LDL reduction with Alirocumab vs 20% reduction with ezetimibe at 24 wk [bib_ref] Efficacy and safety of alirocumab, a fully human PCSK9 monoclonal antibody, in..., Colhoun [/bib_ref]. OPTIONS Ⅰ trial [bib_ref] Alirocumab as Add-On to Atorvastatin Versus Other Lipid Treatment Strategies: ODYSSEY OPTIONS..., Bays [/bib_ref] patients intolerant to statins (GAUSS-2 and GAUSS-3); standalone in hyperlipidemia (MENDEL-2); heterozygous familial hypercholesterolemia (RUTHERFORD-2 and TAUSSIG); homozygous familial hypercholesterolemia (TESLA and TAUSSIG); with primary hyperlipidemia or mixed cholesterol disorder (THOMAS-1 and THOMAS-2: Device trials). Also, long-term safety and efficacy data is being evaluated by the five following studies: DESCARTES; FOURIER; OSLER-2 trial; GLAGOV trial and TAUSSIG study.
In LAPLACE-2 study 1896 patients with fasting LDL ≥ 150 (when not on statins) or LDL ≥ 100 (on nonintense regimen of statins) or LDL ≥ 70 (on intensive statin therapy) were randomized to a daily moderate or high-intensity statin regimen and after 4 wk, further randomized to receive Evolocumab, ezetimibe or placebo. Evolocumab was shown to reduce LDL levels by 66% to 75% (on every 2 wk regimen) and by 63% to 75% (on once monthly regimen) when compared to placebo in moderate-and high intensity statin groups. In moderate and high intensity statin groups, Evolocumab led to significant reduction in absolute LDL values in both regimens of Evolocumab (every 2 wk and monthly). Adverse events reported were comparable in all groups [bib_ref] Effect of evolocumab or ezetimibe added to moderateor high-intensity statin therapy on..., Robinson [/bib_ref]. YUKAWA-2 study showed a similar reduction in LDL in 404 Japanese patients when Evolocumab regimens (140 mg once every 2 wk and 420 mg once a month) were compared to placebo on 2 regimens of low-dose background statin therapy (5 mg/d and 20 mg/d atorvastatin). Interestingly, the reduction in LDL appeared to be similar irrespective of statin dosage (in combination with Evolocumab) and showed a 67% to 76% LDL reduction at 12 wk [bib_ref] Effects of evolocumab (AMG 145) in hypercholesterolemic, statin-treated, japanese patients at high..., Kiyosue [/bib_ref]. MENDEL-2 trial compared the efficacy of Evolocumab with ezetimibe and placebo in 614 patients with LDL between 100 mg/dL and 190 mg/dL and low risk on Framingham scale (≤ 10%). Evolocumab was shown to reduce LDL by up to 57% more than placebo and 40% more than ezetimibe after 12 wk of therapy [bib_ref] Anti-PCSK9 monotherapy for hypercholesterolemia: the MENDEL-2 randomized, controlled phase III clinical trial..., Koren [/bib_ref]. GAUSS-2 trial evaluated 307 patients with statin intolerance and compared the 2 regimens of Evolocumab (140 mg once every 2 wk and 420 mg once a month) to daily oral or subcutaneous placebo (both placebo groups on ezetimibe). At 12 wk, Evolocumab group showed a reduction in LDL by 56% vs 39% in the other groups (placebo + ezetimibe arm) [bib_ref] Anti-PCSK9 antibody effectively lowers cholesterol in patients with statin intolerance: the GAUSS-2..., Stroes [/bib_ref]. Along similar lines, GAUSS-3 trial evaluated the efficacy of Evolocumab in 218 statin intolerant patients compared to ezetimibe. The initial phase of the study included administration of atorvastatin (20 mg) for 10 wk and placebo randomized in a 1:1 fashion, followed by a 2-wk washout period and crossover to alternate therapy for another 10 wk. The patients who experienced muscle related adverse effects while on statin therapy and not on placebo were further enrolled in the second phase of the study, which was a 24 wk double blinded randomized controlled trial to compare Evolocumab (420 mg/mo divided in 3 doses) with ezetimibe (10 mg/d). At 24 wk, LDL-C was hoc analysis, the reduction in LDL was also associated with reduction in the combined end-point of death from coronary artery disease, nonfatal myocardial infarction, fatal or nonfatal ischemic stroke or unstable angina requiring hospitalization (1.7% with Alirocumab vs 3.3% with placebo; HR = 0.52; 95%CI: 0.31-0.9; P = 0.02) [bib_ref] Efficacy and safety of alirocumab in reducing lipids and cardiovascular events, Robinson [/bib_ref]. The ODYSSEY Outcomes trial (NCT01663402) is ongoing is closed to recruitment, and will assess the effects of Alirocumab on CVD events in 18000 patients on maximally tolerated statin therapy. Results of this trial are expected in February 2018.
## Evolocumab (status: fda approved)
Evolocumab has been studied in two phase Ⅰ studies. Dias et al [bib_ref] Effects of AMG 145 on low-density lipoprotein cholesterol levels: results from 2..., Dias [/bib_ref] evaluated healthy volunteers in phase Ⅰa and showed a short-term dose-dependent reduction in LDL by up to 65% and after 6 to 8 wk of therapy by up to 75% with a maximally administered dose of 420 mg subcutaneously/intravenously. Phase Ⅰb trial similarly demonstrated up to 75% reduction in LDL as compared to placebo over 1 to 4 wk in healthy volunteers.
Four phase Ⅱ trials were subsequently performed that continued to show the benefits of Evolocumab with a dose-dependent reduction of LDL (maximal dosing up to 420 mg) when added to maximally tolerated statin therapy in patients with hypercholesterolemia (including familial hypercholesterolemia) [bib_ref] Efficacy, safety, and tolerability of a monoclonal antibody to proprotein convertase subtilisin/kexin..., Giugliano [/bib_ref] [bib_ref] Efficacy, safety, and tolerability of a monoclonal antibody to proprotein convertase subtilisin/kexin..., Koren [/bib_ref] [bib_ref] Low-density lipoprotein cholesterol-lowering effects of AMG 145, a monoclonal antibody to proprotein..., Raal [/bib_ref]. In LAPLACE-TIMI57 trial [bib_ref] Efficacy, safety, and tolerability of a monoclonal antibody to proprotein convertase subtilisin/kexin..., Giugliano [/bib_ref] , Evolocumab was tested at varying doses ranging from 70 to 140 mg every 2 wk or 280 to 420 mg every 4 wk in 631 patients on stable statin therapy and LDL more than 85 mg/dL. LDL reduction up to 65% was observed with the every two-week regimen as compared to approximately 50% LDL reduction with the every 4-wk regimen. Evolocumab has also been studied as monotherapy in 160 patients with hypercholesterolemia and intolerance to statins in the GAUSS trial [bib_ref] Effect of a monoclonal antibody to PCSK9 on low-density lipoprotein cholesterol levels..., Sullivan [/bib_ref]. At doses of 420 mg every 4 wk, it was shown to reduce LDL by 40% to 50%. Furthermore, addition of ezetimibe reduced LDL by up to 65%. Subsequently, the MENDEL trial [bib_ref] Efficacy, safety, and tolerability of a monoclonal antibody to proprotein convertase subtilisin/kexin..., Koren [/bib_ref] showed a similar efficacy in LDL-C lowering when
Evolocumab was used as monotherapy in 406 patients with hypercholesterolemia. Based on these trials, the optimal frequency of Evolocumab therapy was determined to be twice monthly to achieve a 50% to 60% reduction in LDL in combination with statins. However, when used as a monotherapy therapy, a frequency of once every 4 wk would be acceptable. Stein et al [bib_ref] Effect of the proprotein convertase subtilisin/kexin 9 monoclonal antibody, AMG 145, in..., Stein [/bib_ref] evaluated 8 patients with homozygous familial hypercholesterolemia, and found Evolocumab (at 420 mg every 2 wk) to reduce LDL by approximately 25% vs 20% when used every 4
wk. Evolocumab has further been evaluated in PROFICIO (Program to reduce LDL-C and cardiovascular outcomes following inhibition of PCSK9 in different populations) phase Ⅲ trials [fig_ref] Table 2: Summary of important phase Ⅲ PROFICIO [/fig_ref]. The PROFICIO program includes 14 trials where Evolocumab is being evaluated in patients with hyperlipidemia in combination with statins (LAPLACE-2 and YUKAWA-2); hyperlipidemic in LDL with both regimens: 140 mg every 2 wk led to 59.2% reduction (CI: 53.4% to 65.1%) and 420 mg once a month led to LDL reduction by 61.3% (CI: 53.6% to 69%) as compared to placebo after 12 wk (P < 0.001 for all) [bib_ref] PCSK9 inhibition with evolocumab (AMG 145) in heterozygous familial hypercholesterolaemia (RUTHERFORD-2): a..., Raal [/bib_ref]. The TESLA trial examined 50 patients with homozygous familial hypercholesterolemia on stable lipid lowering therapy and evaluated monthly Evolocumab (420 mg) vs placebo therapy for 12 wk. Addition of Evolocumab led to a significant reduction in LDL-C by up to 31% (CI: -44% to -18%; P < 0.0001) [bib_ref] Inhibition of PCSK9 with evolocumab in homozygous familial hypercholesterolaemia (TESLA Part B):..., Raal [/bib_ref].
In the Open-Label Study of Long-Term Evaluation Against LDL-C (OSLER) 1 and 2 trials 4465 patients were enrolled who had completed 1 of the phase 2 or phase 3 studies of Evolocumab (MENDEL-1, LAPLACE TIMI 57, GAUSS-1, RUTHERFORD-1, YUKAWA-1, MENDEL-2, LAPLACE-2, GAUSS-2, RUTHERFORD-2, DESCARTES, THOMAS-1 or THOMAS-2) and randomized to receive either Evolocumab (420 mg/mo in OSLER-1 and 140 mg every 2 wk or 420 mg once a month in OSLER-2 trial) plus standard therapy (n = 2976) or standard therapy (n = 1489). The median follow-up was 11.1 mo. This study showed a 61% reduction in LDL-C with Evolocumab compared to standard therapy (95%CI: 59% to 63%; P < 0.001). Overall adverse events were in 69% of patients in Evolocumab group compared to 65% in standard therapy group. Of note, the neurocognitive adverse events were low, but were more frequent in Evolocumab group and appeared reduced by 53% with Evolocumab compared to 17% with ezetimibe. Muscle-related side effects were reported in 21% patients on Evolocumab compared to 29% with ezetimibe with stoppage of drug administration due to muscle symptoms in 1% of patients in Evolocumab and 7% of patients on ezetimibe [bib_ref] Efficacy and Tolerability of Evolocumab vs ongoing stable atorvastatin therapy, Nissen [/bib_ref]. DESCARTES trial [bib_ref] A 52-week placebocontrolled trial of evolocumab in hyperlipidemia, Blom [/bib_ref] evaluated 901 patients with hyperlipidemia, comparing Evolocumab (420 mg once a month subcutaneous) plus background lipid lowering therapy vs placebo plus background lipid lowering therapy for a period of 52 wk. Background lipid lowering therapy included: Diet alone, low intensity atorvastatin (10 mg), high intensity atorvastatin (80 mg) or atorvastatin 80 mg/d. All patients had fasting LDL-C > 75 mg/dL on background lipid lowering therapy. Addition of Evolocumab resulted in LDL reduction by 51% to 60% in diet alone group, 59% to 64% in patients on 10 mg atorvastatin, 51% to 62% in patients on 80 mg atorvastatin and 43% to 54% in patients with atorvastatin 80 mg/d and ezetimibe 10 mg/d (P < 0.001 for all). Evolocumab has also been shown to be efficacious in patients with heterozygous and homozygous familial hypercholesterolemia. In the RUTHERFORD-2 trial, 329 patients with heterozygous familial hypercholesterolemia were randomized to receive Evolocumab (140 and 420 mg respectively) or placebo at two weekly and monthly regimens. Evolocumab showed a significant reduction TESLA (NCT01588496) Raal et al [bib_ref] Inhibition of PCSK9 with evolocumab in homozygous familial hypercholesterolaemia (TESLA Part B):..., Raal [/bib_ref] ; Randomized double-blindedHomozygous No data are available in patients with severe renal and hepatic impairment.
## Evolocumab
The pharmacokinetic and pharmacodynamics properties of Evolocumab [bib_ref] Efficacy and safety of longer-term administration of evolocumab (AMG 145) in patients..., Koren [/bib_ref] demonstrate non-linear pharmacokinetics in absorption at doses below 140 mg; however, between doses of 140 to 420 mg linear pharmacokinetics is observed. The time to reach maximum concentration is 3 to 4 d after a single dose. After a single 420 mg dosage, its volume of distribution has been estimated to be 3.3 L ± 0.5 L. A steady state in serum is observed after about 12 wk of dosing. The t1/2 of Evolocumab is between 11 to 17 d. The maximum reduction of LDL after therapy was similar after dosing of 140 mg every 2 wk or 420 mg once a month with effect within 14 d. Clinical studies have not revealed a difference in pharmacokinetics of Evolocumab in mild or moderate renal and hepatic impairment. However, subjects with severe renal and hepatic impairment have not been studied.
## Adverse effects and contraindications
The following side effects have been reported by data gathered from over 7000 patients (n = 2476 for Alirocumab and n = 5416 for Evolocumab) evaluated in the clinical trials mentioned above. Major side effects observed for Alirocumab and Evolocumab are described below.
## Alirocumab
Alirocumab is contraindicated in patients who develop serious hypersensitivity reactions like hypersensitivity vasculitis or allergic reactions requiring hospitalization with its usage. The most common adverse effects observed with Alirocumab include nasopharyngitis (11.3% vs 11.1% in placebo), injection site reactions (erythema, itchiness, swelling, pain or tenderness) [bib_ref] Efficacy and safety of longer-term administration of evolocumab (AMG 145) in patients..., Koren [/bib_ref] [bib_ref] Efficacy and safety of evolocumab in reducing lipids and cardiovascular events, Sabatine [/bib_ref]. The TAUSSIG trial (NCT01624142) is evaluating Evolocumab therapy in 300 patients with severe familial hypercholesterolemia to determine its efficacy and side effect profile. The results of this study are anticipated by March 2020. Preliminary results reported by Stein et al [bib_ref] Effect of the proprotein convertase subtilisin/kexin 9 monoclonal antibody, AMG 145, in..., Stein [/bib_ref] on 8 patients with LDLR-negative or LDLR defective homozygous familial hypercholesterolemia on stable drug therapy when treated with Evolocumab at 420 mg monthly for ≥ 12 wk, followed by 420 mg every 2 wk for another 12 wk showed LDL reduction by 14% to 16% at 12 wk with 2 wk and 4 wk dosing regimens respectively with no serious adverse events reported [bib_ref] Effect of evolocumab or ezetimibe added to moderateor high-intensity statin therapy on..., Robinson [/bib_ref]. Finally, the preliminary results of GLAGOV study (NCT01813422) evaluating 950 patients with coronary artery disease on lipid lowering therapy undergoing cardiac catheterization for changes in percentage atheroma volume after 78 wk of Evolocumab therapy met primary and secondary endpoints and final results are to be reported in American Heart Association (AHA) conference in November, 2016.
## Pharmacokinetics and
## Pharmacodynamics
The pharmacokinetic and pharmacodynamics parameters of PCSK9 inhibitors are described below [bib_ref] A randomized study of the relative pharmacokinetics, pharmacodynamics, and safety of alirocumab,..., Lunven [/bib_ref] [bib_ref] Profile of evolocumab and its potential in the treatment of hyperlipidemia, Cicero [/bib_ref].
## Alirocumab
The time taken to reach maximum serum concentration is 3-7 d with similar serum concentration -time profiles between abdomen, upper arm or thigh as the sites of injection. Steady state concentrations are reached at an average of 3 to 4 doses. The volume of distribution following intravenous administration is 0.04 to 0.05 L/kg. The median half-life (t1/2) observed was between 17 to 20 d at 75 or 150 mg dosing every 2 wk. Alirocumab is eliminated in two phases depending upon its plasma concentration. The predominant mode of elimination at lower concentrations is via saturation of the targets (PCSK9) bound to the antibodies; however, at higher concentrations it is primarily through proteolytic pathways [bib_ref] PCSK9 inhibition with evolocumab (AMG 145) in heterozygous familial hypercholesterolaemia (RUTHERFORD-2): a..., Raal [/bib_ref]. There have been no metabolism studies conducted since it has been previously demonstrated that reticuloendothelial system is responsible for metabolizing antibodies to small peptides and amino acids [bib_ref] Metabolism of immunoglobulins, Waldmann [/bib_ref]. The maximum reduction in free plasma PCSK9 levels and LDL was observed within 3 and 15 d respectively, after drug administration with no difference noted between different sites. No dose adjustment is needed for patients with mild or moderately impaired renal or hepatic function. PCSK9 inhibitors are especially beneficial in the treatment of familial hypercholesterolemic patients who are intolerant to statins or have an elevated LDL-C level despite being on maximally tolerated statin therapy. Intuitively, addition of a PCSK9 inhibitor to low dose statin therapy will be more effective in lowering LDL and avoiding the side effects of statins, since low dose and high dose statin regimens have yielded similar efficacy when combined with PCSK9 inhibitors.
Several potential barriers exist that may impede the widespread use of these medicines. First, statins have a proven effectiveness that has been demonstrated in multiple long-term studies. Statins have been shown to reduce cardiovascular mortality by 30% and incidence of stroke by 20% in multiple long-term studies [bib_ref] Statins for primary prevention of cardiovascular disease: the benefits outweigh the risks, Minder [/bib_ref]. PCSK9 inhibitors are effective in reducing LDL-C levels but currently lack data demonstrating their use reduces CVD events. Trials evaluating the effect of PCSK9 inhibitors on long-term CVD events, however, are currently underway: FOURIER (Further cardiovascular outcomes research with PCSK9 inhibition in subjects with elevated risk; n = 22500) for Evolocumab (NCT01764633) and ODYSSEY-OUTCOMES (ODYSSEY outcomes: Evaluation of cardiovascular outcomes after an acute coronary syndrome during treatment with alirocumab SAR236553) (NCT01663402) for Alirocumab. However, their data will not be available until December 2017 (for Alirocumab) and February 2018 (for Evolocumab).
Another potential barrier to widespread use of PCSK9 inhibitors is their cost. The Institute for Clinical and Economic Review (ICER) reported that the number needed to treat for 5 years to avoid one major adverse cardiovascular event (NNT5) is 28. However, a list price of $ 14350 per year generates a cost-effectiveness ratio which far exceeds the accepted threshold of $ 100000/quality-affected life-years [bib_ref] ACC/AHA statement on cost/ value methodology in clinical practice guidelines and performance..., Anderson [/bib_ref]. To achieve costeffectiveness at this threshold would require a price reduction by 60% to 65% of the current price. At the conclusion of their report, the ICER suggested a reduction by 85% to an annual cost of $2177 might be required to avoid excessive cost burdens to the health care system. It should be noted that since there are limited data on clinical adverse cardiovascular events, cost effectiveness data might change once results from ongoing CVD endpoint studies are available.
PCSK9 therapy is a welcome treatment option for statin intolerant patients who require treatment of their hyperlipidemia. It will be important that busy practitioners do not under-prescribe statins nor be dissuaded from attempting to find a dose of and statin agent that is tolerated by the patient because PCSK9 inhibitors are available. Despite these obstacles, PCSK9 inhibitors are an exciting agent for reducing LDL-C hyperlipidemia and have ushered in a new era of lipid lowering therapy. in placebo), back pain (3% vs 2.7% in placebo) and nausea (2.1% vs 1.8% in placebo). Of note the most common adverse events leading to drug discontinuation include myalgia, nausea and dizziness. Other serious adverse events noted were cardiac disorders in 2.4% individuals including palpitations (0.6% vs 0.3% in placebo), angina pectoris (0.3% vs 0.2% in placebo), and ventricular extra systoles (0.3% vs 0.1% in placebo).
In addition, data from trials evaluating Evolocumab and Alirocumab have shown a higher incidence of cognitive adverse events in patients treated with PCSK9 inhibitors (0.9% vs 0.3% for Evolocumab compared to standard care [bib_ref] Efficacy and safety of longer-term administration of evolocumab (AMG 145) in patients..., Koren [/bib_ref] and 1.2% vs 0.5% for Alirocumab compared to placebo [bib_ref] Effects of AMG 145 on low-density lipoprotein cholesterol levels: results from 2..., Dias [/bib_ref]. It has been suggested that responder and ascertainment bias might have played a role in reporting of adverse cognitive events in the OSLER trial since the adverse events were not related to the degree of LDL-C lowering with no clustering in the LDL-C < 25 mg/dL group relative to the 25-50 mg/dL or > 50 mg/dL groups. However, patients in ODYSSEY LONG TERM trial were blinded to treatment and followed for nearly 18 mo. Also, the neurocognitive adverse events were measured subjectively and not verified by neurocognitive testing. A dedicated study evaluating neurocognitive adverse events with PCSK9 inhibitors is underway: Evaluating PCSK9 Binding anti-Body Influence oN coGnitive HeAlth in High cardiovascUlar Risk Subjects (EBBINGHAUS) (NCT02207634). It is enrolling individuals without dementia or mild cognitive impairment at baseline randomized in a double-blind, placebo-controlled fashion to evaluate Evolocumab on background statin therapy vs statin therapy alone. The primary outcome being measured is Spatial Working Memory test; an assessment of executive function and the results are expected in September 2017.
Another potential and significant complication with drugs that are monoclonal antibodies is the development of anti-drug antibodies that may interfere with clinical efficacy and increase adverse events [bib_ref] The safety of therapeutic monoclonal antibodies: implications for cardiovascular disease and targeting..., Catapano [/bib_ref]. This complication has not been reported in the trials to date using PCSK9 inhibitors.
## Clinical use of pcsk9 inhibitors
The 2013 American College of Cardiology/American Heart Association recommendations on cholesterol management centered on identifying patients who would have a reduction in CVD events with statin treatment. The focus shifted from treating to a specific LDL-C level, to treating at risk individuals with a treatment (statin) proven to reduce future CVD events. Subsequently, the IMPROVE-IT trial demonstrated that addition of ezetimibe to simvastatin lowers LDL-C more than that achieved by simvastatin alone and that this reduction in LDL-C was associated with a greater reduction in CVD events compared with simvastatin alone [bib_ref] Profile of evolocumab and its potential in the treatment of hyperlipidemia, Cicero [/bib_ref] [bib_ref] Proof That Lower Is Better--LDL Cholesterol and IMPROVE-IT, Jarcho [/bib_ref]. This study raises the issue of LDL-C treatment targets with a lower level of LDL-C corresponding to a lower risk of CVD events. The introduction of PCSK9 inhibitors will necessitate re-evaluation of existing cholesterol treatment recom-Chaudhary R et al . Review on PCSK9 inhibitors
[fig] Figure 1: Mechanism and role of PCK9 in low-density lipoprotein-cholesterol metabolism. LDL: Low-density lipoprotein. [/fig]
[table] Table 2: Summary of important phase Ⅲ PROFICIO (Program to Reduce LDL-C and Cardiovascular Outcomes Following Inhibition of PCSK9 In Different Populations) trials with Evolocumab [/table]
|
Synthetic DNA and RNA Programming
Synthetic biology is a broad and emerging discipline that capitalizes on recent advances in molecular biology, genetics, protein and RNA engineering as well as omics technologies. Together these technologies have transformed our ability to reveal the biology of the cell and the molecular basis of disease. This Special Issue on "Synthetic RNA and DNA Programming" features original research articles and reviews, highlighting novel aspects of basic molecular biology and the molecular mechanisms of disease that were uncovered by the application and development of novel synthetic biology-driven approaches.Synthetic biology is a broad and emerging discipline that capitalizes on recent advances in molecular biology, genetics, protein and RNA engineering, as well as omics technologies. Together these biotechnologies have transformed our ability to reveal the biology of the cell and the molecular basis of disease. This special issue of Genes on "Synthetic DNA and RNA Programming" features 12 original research articles and reviews that highlight novel aspects of basic molecular and cellular biology, uncovered by the application and development of synthetic biology-driven approaches.The approaches highlighted here involve programming genes, RNAs, and proteins, both in the test tube and in living cells, to explore areas of molecular biology related to genetic code evolution[1,2]and genetic code expansion[3][4][5], including applications in programming protein modifications[6,7], RNA metabolism[8,9], and genetic systems for genome engineering[10,11]and biocontainment of modified microorganisms[12]. These contributions showcase both the diversity of research approaches emerging as synthetic biology tools and the extreme level of detail with which these tools enable studies and the manipulation of individual protein modifications or cellular pathways. The contributed articles cluster into four thematic categories: genetic code expansion, genetic code evolution, novel genetic systems and molecular tools, and RNA programming.
## Expanding the genetic code
Genetic code expansion studies focus on methods that enable the production of proteins with amino acids beyond the canonical or standard 20 genetically encoded amino acids. Cells with engineered and expanded genetic codes can produce proteins with 21or even 22different genetically encoded amino acids. These additional amino acids allow the introduction of new and specific chemically functionalized side chains.
Genetic code expansion normally requires the addition of a new aminoacyl-tRNA synthetase and tRNA pair. Efficient and high-fidelity genetic encoding requires that the AARS tRNA pair is mutually orthogonal to the endogenous AARSs and tRNAs in the cell. These methods normally reassign the meaning of a stop codon, usually UAG, to the direct incorporation of an additional, non-canonical amino acid (ncAA). The most commonly used orthogonal pairs include the archaeal enzymes tyrosyl-tRNA syntetase (TyrRS), the pyrrolysyl-tRNA synthetase (PylRS), and the phosphoseryl-tRNA synthetase (SepRS). Balasuriya et al. used the phosphoserine system to produce highly active human kinases with programmed phosphorylation from facile Escherichia coli expression systems. The authors used these reagents to identify a specific phosphorylation site on protein kinase B (Akt1) that interferes with a clinically relevant kinase inhibitor.
Genetic code expansion also enables the incorporation of other posttranslational modifications at specific or programmed sites in proteins. Umehara et al.used a mutant of the PylRS tRNA Pyl orthogonal pair to genetically encode N ε -acetylated lysine into the E. coli alanyl-tRNA synthetase. The resulting acetylated AlaRS was catalytically deficient. The authors next used in vivo assays to determine that two Cob deacetylases were able to remove the K73 acetylation, identifying a novel regulatory pathway to control AlaRS activity.
The study from Hoffmann et al. focused on the central role of tRNAs in genetic code expansion. Specifically, this review article addressed two 'fundamentally different translation systems' that evolved in natural organisms. These include the systems that genetically encode selenocysteine and pyrrolysine, the so-called 21st and 22nd amino acids. Design and engineering of tRNA variants for optimal genetic code expansion were examined, as were synthetic biology applications of genetic code expansion.
Finally, Gang et al. focused on the application of peptides with ncAAs as antibiotics. Antibiotic development is a matter of urgent clinical need and one of the most promising areas is related to the development of synthetic cyclic peptides. These compounds are often inspired by natural products, but through the use of genetic code expansion, ncAAs can be included in these antibiotic peptides. The review article from Gang et al. focused on methods for peptide cyclization and applications of cyclic peptides.
## Genetic code evolution
Although Crick's frozen accident hypothesis once envisioned a perfectly interpreted genetic code, recent studies have found that cells tolerate surprisingly high levels of amino acid misincorporation. The next pair of featured articlesinvestigated biochemical mechanisms that regulate translation fidelity.
Berg et al.provided the first detailed characterization of human tRNA Ser identity elements. These are the critical nucleotides that are essential for tRNA recognition by the cognate aminoacyl-tRNA synthetase and determine the serine accepting identity of this tRNA. The tRNA identity elements define the correspondence of amino acids with anticodons and thus are fundamental to the accurate decoding of the genetic code. Using a unique in vivo assay, relying on the misincorporation of serine at proline codons in yeast, this work identified the evolution of new identity elements in the human tRNA Ser .
Similarly, the article by Rathnayake et al.also highlighted the fact that the mechanisms and molecular entities tasked with ensuring the faithful interpretation of genetic code have evolved over time into idiosyncratic variants. The aspartyl-tRNA synthetase (AspRS) enzymes occur as two variants, the discriminating AspRS, which exclusively charges Asp to tRNA Asp , as well as a non-discriminating variant (ND-AspRS). ND-AspRS charges Asp onto tRNA Asn in a pathway generating Asn-tRNA Asn via Asp-tRNA Asn . In some bacteria, both AspRS and ND-AspRS are capable of not only charging Asp to tRNA Asp and tRNA Asn , but also of have an unexpected glutamyl-tRNA synthetase (GluRS)-like activity. In these cases, AspRS acts as a GluRS, and is able to ligate Glu to tRNA Glu but not to tRNA Asp . Although the AspRS enzyme targets a non-cognate tRNA, amino acid misincorporation does not occur, thus preserving translation fidelity.
## Novel genetic systems and molecular tools
Gordon et al. opened the door to studying the unusual biology of yeast of the genus Metschnikowia. These yeasts have a genetic code variation in which the leucine CUG codons are instead decoded as serine. The authors developed a new and efficient transformation system for Metschnikowia and an additional 21 yeast species, providing new platforms for genome synthesis and engineering efforts.
Protein expression is an essential methodology for biochemical studies, including those mentioned above, as well as for the production of biopharmaceuticals such as antibodies and therapeutic peptides. Chen et al.used a high-throughput approach to screen for the impact of the expression of any E. coli gene on cell growth. The most significant impact on E. coli growth was observed upon the overexpression of membrane proteins. Interestingly, the authors found that for certain proteins, the use of lower copy number plasmids stabilized cell growth rate and increased overall recombinant protein yields. The impact on cell growth could also often be remedied by amino acid supplementation. Using a related methodology, Schwark et al. used a fluorescent protein reporter system and investigated an E. coli release factor deletion strain with a highly efficient archaeal orthogonal AARS tRNA pair. Their data suggested that the engineered strain will be a highly useful tool for applications requiring the incorporation of multiple ncAAs.
A major ethical responsibility for synthetic biologists is the ability to retain control or confinement of genetically modified microorganisms. This drove the field to develop fascinating and effective methods for containing microorganisms in the lab that are commonly referred to as biocontainment. Some of these approaches included the use of genetic code expansion to create synthetic auxotrophs, such as an E. coli strain dependent on ncAAs for growth. Diwo et al. further extended this idea with their novel formulation of an 'alien genetic code'. The authors presented the concept that an ideal biocontainment system would involve the creation of microbes with genetic codes that are totally incompatible or 'alien' to the natural genetic code. The construction of such an organism is considered to be achieved via the directed evolution of an existing cell or through de novo construction of 'synthetic' genomes and cells.
## Rna programming
RNAs play important roles in controlling translation, yet there are still limited tools to study and engineer RNAs and their activity in cells. Turk et al. developed a novel reporter system to quantify the activity of microRNAs (miRNAs) in living cells. While microRNAs can be quantified by methods such as quantitative PCR, a variable and unknown portion of cellular miRNAs is inactive. To assess the amount of active miRNA in the cell, the authors developed a GFP-based reporter system that allows for time-and space-resolved monitoring of active miRNAs in single cells.
Another limitation of analyzing RNAs is the limitation of commercially available polymerases to extend RNAs in the forward 5 to 3 direction. Chen et al.reviewed recent progress made towards the characterization and engineering of tRNA His guanylyltransferase homologs capable of reverse 3 to 5 nucleotide polymerization. Tools and enzymes with broadened RNA substrates and extended template-dependent reverse polymerization activity were discussed and will be invaluable for RNA labeling, engineering, and analysis efforts.
In summary, this collection of articles represents new directions in multiple areas of interest to synthetic biologists. From expanding the number of genetically encoded amino acids to creating new genetic tools in diverse microbes, these articles are each exemplars of the sophisticated synthetic biology approaches that produce cells with new capabilities, enabling the production of designer proteins and RNAs. Some of these tools reveal molecular events in living cells that were previously inaccessible. |
Intrinsic Functional Brain Architecture Derived from Graph Theoretical Analysis in the Human Fetus
The human brain undergoes dramatic maturational changes during late stages of fetal and early postnatal life. The importance of this period to the establishment of healthy neural connectivity is apparent in the high incidence of neural injury in preterm infants, in whom untimely exposure to ex-uterine factors interrupts neural connectivity. Though the relevance of this period to human neuroscience is apparent, little is known about functional neural networks in human fetal life. Here, we apply graph theoretical analysis to examine human fetal brain connectivity. Utilizing resting state functional magnetic resonance imaging (fMRI) data from 33 healthy human fetuses, 19 to 39 weeks gestational age (GA), our analyses reveal that the human fetal brain has modular organization and modules overlap functional systems observed postnatally. Age-related differences between younger (GA ,31 weeks) and older (GA$31 weeks) fetuses demonstrate that brain modularity decreases, and connectivity of the posterior cingulate to other brain networks becomes more negative, with advancing GA. By mimicking functional principles observed postnatally, these results support early emerging capacity for information processing in the human fetal brain. Current technical limitations, as well as the potential for fetal fMRI to one day produce major discoveries about fetal origins or antecedents of neural injury or disease are discussed.
# Introduction
A major objective for neuroscience is to build a complete diagram of brain connections at the beginning of human life. Functional MRI has recently proven capable of measuring neural connectivity in the human fetal brain [bib_ref] Watching the fetal brain at 'rest', Schöpf [/bib_ref] [bib_ref] Cross-hemispheric functional connectivity in the human fetal brain, Thomason [/bib_ref]. By leveraging correlations of low-frequency (,,0.1 Hz) intrinsic fluctuations in the blood oxygen level dependent (BOLD) signal, functional connectivity MRI (fcMRI) provides information about macroscale brain organization. While this method is based on functional signals, intrinsic brain fluctuations have been shown to reflect underlying anatomic pathways [bib_ref] Loss of resting interhemispheric functional connectivity after complete section of the corpus..., Johnston [/bib_ref] [bib_ref] Intrinsic functional architecture in the anaesthetized monkey brain, Vincent [/bib_ref] , making this a useful technique for exploring emergent neural circuits in the human fetus.
Graph theory is a method used in mathematics to extract global organizational principles for physical, biological, or social systems by modeling interrelations between units or members of the system. For example, airline flight routes, migration patterns, social networks, and Twitter feeds all may be studied using graph theory. This technique conveys information about overall network infrastructure as well as specific features, such as which 'nodes' (locations/individuals) within a system are central 'hubs' of connectivity, linking numerous other units to one another. In the past several years, graph theory-based approaches have proven highly effective for defining organizational structure of human brain networks (reviewed by [bib_ref] Graph-based network analysis of resting-state functional MRI, Wang [/bib_ref]. For example, from graph analysis of fMRI datasets we have learned that the human brain is organized with small world topology [bib_ref] Normalized cut group clustering of resting-state FMRI data, Van Den Heuvel [/bib_ref] and that the posterior cingulate and insular cortices are connectivity hubs [bib_ref] The precuneus/posterior cingulate cortex plays a pivotal role in the default mode..., Fransson [/bib_ref] [bib_ref] Mapping the structural core of human cerebral cortex, Hagmann [/bib_ref] [bib_ref] Precuneus shares intrinsic functional architecture in humans and monkeys, Margulies [/bib_ref].
Graph analysis of fMRI data involves first dividing the brain into a set of distinct predefined regions from which BOLD timeseries are extracted, then correlating timeseries with one another in a pairwise fashion, yielding an n6n correlation matrix. Here, each a priori selected brain region, or n, is a 'node', while each matrix value is an 'edge' denoting the relationship for a given pair. Edges are reported either as weighted with positive/negative connection strength above a threshold, or as binary . Diverse metrics exist for gleaning topological network organization from brain connectivity matrices (see [bib_ref] Graph-based network analysis of resting-state functional MRI, Wang [/bib_ref] , but it has been suggested that when numerous edges within the graph are negative, modular assessment is a preferred approach [bib_ref] Weight-conserving characterization of complex functional brain networks, Rubinov [/bib_ref].
Modularity measures the degree to which a network can be partitioned into non-overlapping subsets of regions (''modules'') that are internally interactive, while sparsely interactive with outside areas. Within a modular network, critical nodes (brain regions) serve specific roles integrating local connections (intramodular hubs) or branching between modules (intermodular connectors [bib_ref] Hierarchical modularity in human brain functional networks, Meunier [/bib_ref]. First applied to adults [bib_ref] A resilient, low-frequency, small-world human brain functional network with highly connected association..., Achard [/bib_ref] [bib_ref] Scale-free brain functional networks, Eguiluz [/bib_ref] [bib_ref] Undirected graphs of frequency-dependent functional connectivity in whole brain networks, Salvador [/bib_ref] , and later to children [bib_ref] Development of distinct control networks through segregation and integration, Fair [/bib_ref] [bib_ref] Development of Large-Scale Functional Brain Networks in Children, Supekar [/bib_ref] and infants [bib_ref] The functional architecture of the infant brain as revealed by resting-state fMRI, Fransson [/bib_ref] [bib_ref] Evidence on the emergence of the brain's default network from 2-week-old to..., Gao [/bib_ref] , graph-based fMRI analyses show that the human brain has modular architecture.
Prior work has highlighted age-related differences in module size and composition [e.g., [bib_ref] Development of Human Brain Structural Networks Through Infancy and Childhood, Huang [/bib_ref] [bib_ref] Age-related changes in modular organization of human brain functional networks, Meunier [/bib_ref] demonstrating the utility of tracing the profile of modular architecture across development. As children mature, brain network configuration shifts from a local to a distributed organization. That is, characteristic functional brain subnetworks or ''modules'' emerge wherein spatially distant brain regions such as frontal and parietal regions, comprising the executive control network, and between medial prefrontal and parietal regions, comprising the default mode network, become more strongly connected [bib_ref] Functional brain networks develop from a ''local to distributed'' organization, Fair [/bib_ref].
Graph-based MRI analyses are relevant to cognition [bib_ref] Large-scale cortical network properties predict future sound-to-word learning success, Sheppard [/bib_ref] , and can differentiate unique patterns of atypical connectivity in neurodevelopmental disorders such as attention deficit-hyperactivity disorder (ADHD [bib_ref] Distinct neural signatures detected for ADHD subtypes after controlling for micro-movements in..., Fair [/bib_ref] , autism [bib_ref] Using multiparametric data with missing features for learning patterns of pathology, Ingalhalikar [/bib_ref] [bib_ref] Altered functional and structural brain network organization in autism, Rudie [/bib_ref] , and Tourette's syndrome [bib_ref] Functional immaturity of cortico-basal ganglia networks in Gilles de la Tourette syndrome, Worbe [/bib_ref]. Overall, graph analysis has proven valuable in understanding human brain organizational structure, developmental processes, and disease sequelae. However, it is unknown whether recognizable principles of human functional brain organization (i.e. modular organization) are evident before birth. The present study utilizes a graph theoretical analysis to elucidate the functional architecture of the human fetal brain.
# Materials and methods
## Enrollment
Participants were recruited while receiving prenatal care at clinics located in Hutzel Women's Hospital. Physicians provided initial orientation to the study. Interested and eligible women were introduced to members of the research team who provided a study overview. This included introduction to MRI technology, a study timeline, and a review of the consent form. Exclusion criteria included history of claustrophobia or contraindications for MRI. In addition, the topic of safety and fetal MRI was discussed prior to enrollment. For a review of safety and fetal fMRI, please refer to prior works that have addressed this topic in more detail [bib_ref] A review of the current use of magnetic resonance imaging in pregnancy..., De Wilde [/bib_ref] [bib_ref] Fetal Magnetic Resonance Imaging at 3Tesla, Welsh [/bib_ref]. Women were informed that their participation in this research was voluntary and not related to their clinical care. They were assured that they may discontinue involvement at any time and that personal information would remain confidential.
## Participants
Thirty-eight pregnant women (singleton, uncomplicated pregnancies, median maternal age = 24 years, range = 18 to 34 years) underwent MRI examination within the 19 th to 39 th week of pregnancy. The median gestational age (GA) of their fetuses was 31 weeks (range = 19+6 to 38+5, weeks+days) at the time of MRI. Five participants were excluded prior to group level analyses due to fewer than 90 fMRI volumes retained after removing high movement frames, leaving a total of 33 participants. fMRI data from 19 of these participants has been reported previously [bib_ref] Cross-hemispheric functional connectivity in the human fetal brain, Thomason [/bib_ref].
# Ethics statement
Written informed consent to undergo MRI was obtained from all participants. The Human Investigation Committee of Wayne State University approved the study protocol.
## Image acquisition
Participants underwent MRI examination using a Siemens Verio 70-cm open-bore 3-T MR system using a 550 g abdominal 4-Channel Siemens Flex Coil. Resting-state fMRI data were collected with the following echo planar imaging (EPI) BOLD parameters: repetition time/echo time (TR/TE), 2000/30 ms; 4 mm slice thickness, no skip; 25 axial slices; 80u flip angle. Realtime adjustments were made to imaging protocols during scan acquisition; in cases where data were cut off, image artifacts were present, or movement levels were appraised as high, additional BOLD fMRI data were collected. As a result, the total number of fMRI time fames obtained varied from 180 to 463, x = 343, across participants. The total imaging protocol was restricted to a maximum of 45 minutes duration.
## Functional data preprocessing
Fetal data were preprocessed using methods adapted from our prior work [bib_ref] Cross-hemispheric functional connectivity in the human fetal brain, Thomason [/bib_ref]. In brief, preprocessing included manual segmentation of the fetal brain from surrounding tissue, manual reorientation, volume realignment, normalization to a 32-week fetal template [bib_ref] Construction of a consistent high-definition spatio-temporal atlas of the developing brain using..., Serag [/bib_ref] retaining native resolution (3.4463.4464 mm), segment realignment, and smoothing.
## Movement
Multiple steps were performed to address image motion-related artifacts. First, only low movement time frames were included in analyses. This strategy draws on the fact that although motion in the fetus is substantial, there are periods of relative stability (e.g., , 1.5 mm) for which conventional volume motion correction would be expected to suffice. Participants retaining fewer than 90 volumes after frame exclusion were excluded from analyses. Next, volume realignment was performed using SPM8. Following this, mean framewise displacement across the scan and root mean square (RMS) were calculated and averaged for translational (T x , T y , T z ) and rotational (R pitch , R roll , R yaw ) movement. Lastly, translational and rotational movement parameters (with another six parameters representing their first order temporal derivatives) were removed with covariate regression analysis before computing ROI pairwise correlations. Two-sample t-tests were used to compare translational and rotational movement parameters between younger and older fetal groups (younger, GA ,31 weeks, n = 17; older GA$31 weeks; n = 16). Any movement parameter that was found to be significantly different between age groups was included as covariate in partial correlations assessing correspondence between age and graph theoretical measures. Movement data were processed with SPSS version 21, using a p#0.05 significance level.
## Spectral clustering for region of interest (roi) identification
A spatially constrained group level clustering approach [bib_ref] A whole brain fMRI atlas generated via spatially constrained spectral clustering, Craddock [/bib_ref] was used to generate spatially contiguous ROIs that are evenly sized and distributed across the cortex, including the cerebellum, and excluding cerebral spinal fluid (CSF). Briefly, this method produces functionally homogenous clusters by assessing voxel timeseries similarity using Pearson correlations, then iteratively merges voxels whose within-cluster similarity is maximal and between-cluster similarity is minimal. Next, it identifies the most representative clusters of voxels using a normalized cut algorithm [bib_ref] Normalized cut group clustering of resting-state FMRI data, Van Den Heuvel [/bib_ref] and performs group level clustering. This method produces ROIs that are optimally functionally homogenous and consistent across individuals. Fetal fMRI data were gray matter masked and analyzed to generate 150 ROIs. These were then classified by hemisphere, by lobe, and by coordinates corresponding to center of mass. One of the 150 ROIs was anomalous (spatially discontiguous) and was removed from analysis. Resultant fetal ROI files are available at www.brainnexus.com for download.
## Participant connectivity matrices
The CONN-fMRI Toolbox ver.12.p [bib_ref] Conn: A functional connectivity toolbox for correlated and anticorrelated brain networks, Whitfield-Gabrieli [/bib_ref] was used to measure correlations between ROIs. Rather than removing the global signal to reduce spurious noise effects, we used the anatomical component correction (aCompCor) method of estimating and removing noise [bib_ref] A component based noise correction method (CompCor) for BOLD and perfusion based..., Behzadi [/bib_ref] [bib_ref] Anticorrelations in resting state networks without global signal regression, Chai [/bib_ref]. Principal components of signals from white matter and cerebral spinal fluid, as well as translational and rotational movement parameters (with another six parameters representing their first order temporal derivatives), were removed with covariate regression analysis (see . Pearson's correlation coefficients were then estimated from time series data for each pair of regions. Fisher's transformation was used to convert coefficients to z-scores to produce FC correlation matrices for each participant. Correlation matrices were entered into network analyses using methods described in the following section.
## Graph processing
The Brain Connectivity Toolbox (https://sites.google.com/ site/bctnet/) was used to compute fundamental graph theory metrics. Graphs were examined in their unthresholded, weighted state with negative weights included using the Rubinov-Sporns algorithm to determine the optimal modular partitioning of each participant's network [bib_ref] Weight-conserving characterization of complex functional brain networks, Rubinov [/bib_ref]. We investigated both global and regional topological properties of brain graphs. At the global level, we assessed modularity (Q), which measures the degree to which each network can be clearly separated into minimally overlapping modules by comparing the ratio of within-module edges (connections) and between-module edges in the observed graph versus the ratio expected by chance. A highly modular network is one in which subsets of nodes have many connections between them and few connections to the remaining nodes in the network. Because the modularity algorithm is heuristic and produces minimally varied partitions from run to run, 1000 iterations were run in order to identify the partition that was most consistently identified across runs. The selected partition was then subjected to a finetuning algorithm to further optimize the modularity [bib_ref] Weight-conserving characterization of complex functional brain networks, Rubinov [/bib_ref]. Pearson's correlations were used to test for associations between GA and modularity (Q) and intermodular mean correlation strength.
We assessed two basic nodal properties: positive connection strength and negative connection strength. We then assessed six nodal properties based on each node's pattern of connections within its own module and to the remaining modules. These were: (1) positive participation (correlation) coefficient, (2) negative participation coefficient, (3) within module positive strength Z score, (4) within module negative strength Z score, (5) between . Fetal fMRI graph processing pipeline. 33 preprocessed fetal fMRI datasets (A) were divided into 149 distinct regions of interest (B), from which regional blood oxygen level dependent (BOLD) timeseries was extracted (C). After removing principal components of signals from cerebrospinal fluid (CSF), gray matter (GM), and white matter (WM), as well as the translational and rotational movement parameters (D), timeseries were correlated with one another in pairwise fashion to derive a 1496149 correlation matrix (E), which was used to assess graph metrics (e.g., modularity) and to derive graph visualizations of human fetal brain networks. doi:10.1371/journal.pone.0094423.g001 module positive strength Z score, and (6) between module negative degree Z score. Participation coefficients reflect the fraction of a node's edges that connect to other nodes within the same module. Within module strength z-score is calculated as the summed weight of positive edges connecting a node to other nodes within the same module. This value, the within-module strength, is then scaled by the mean within-module strength for all other nodes in the same module to obtain the corresponding z-score. Between module strength z-scores are calculated in the exact same fashion, except only counting edges between a node and all other nodes belonging to other modules. Robust regression tested the relationship of age and each nodal measure. Fetuses were divided at median age 31 weeks to facilitate comparison of modularity structure in older versus younger fetal groups. P values were corrected for multiple comparisons using false discovery rate correction with p#0.05 (as described by [bib_ref] Controlling the False Discovery Rate: A Practical and Powerful Approach to Multiple..., Benjamni [/bib_ref].
Next, we evaluated the relation between length and strength of connections across ROI pairs. For each participant, correlation strengths obtained for every pairwise comparison (N = ,11,000) were organized into 20 bins of size.1, covering the full range from 21 to 1. Euclidean distance between each ROI pair was computed. Average connection length in each correlation strength bin was determined for each participant. Two sample t-tests were used to compare the strength of the 5% longest connections and 5% shortest connections between older and younger fetuses using an alpha value of p = 0.025.
# Results
Birth outcomes for newborns that were scanned in utero as participants in this study are provided in [fig_ref] Table 1: Summary of Participant and Data Characteristics [/fig_ref].
## Data summary
On average 343 frames, or 11.6 minutes of fMRI data, were collected for the 33 participants included in our analysis. After removing high movement frames, we retained x = 208 frames for each participant, or 60.6% of the total data collected. A larger number of fMRI frames were removed in younger compared to older fetuses (younger x = 168, SD 658; older x = 104, SD 650; p = 0.001), and older fetuses retained a larger number of fMRI frames for analyses (younger x = 187, SD 660; older x = 228, SD 645; p = 0.03). The average duration that fetal data were collected continuously without interruption by movement was 33 consecutive frames, or ,1 minute. The total number of interruptions introduced into consecutive data acquisition by excluding high movement segments was 5.9 (S.D. = 2.1), including breaks between scans. Specific absorption rate (SAR) is a measure of the heating caused by radio-frequency energy deposition in the body. Here, average SAR across fMRI volumes was 0.24 W/kg (S.D. = 0.08), well within FDA safety exposure limits (3 W/kg/10minute exposure).
## Movement
Translational and rotational movement for younger and older fetal groups are summarized in [fig_ref] Table 1: Summary of Participant and Data Characteristics [/fig_ref]. Movement values were on average ,1 millimeter/radian, and thus within accepted standards (cf. [bib_ref] Distinct neural signatures detected for ADHD subtypes after controlling for micro-movements in..., Fair [/bib_ref]. Younger and older fetal groups did not differ in translational or rotational framewise displacement, or RMS, nor did they differ in mean translational movement. However, fetal age groups showed a significant difference in mean rotational movement (p = 0.053), and as a result, mean rotational movement was included as a covariate in all subsequent age-related global analyses.
## Modularity
Intermodular mean connection strength was positively correlated with GA, r = 0.4 p = 0.02. This result indicates that with development, functional modules become more tightly integrated into a whole brain system. There was a related negative relationship between modularity (Q; mean = 0.35, standard deviation = 0.1, range: 0.14-0.6, normally distributed) and GA, r = 20.38 p = 0.03. This suggests that after correction for movement variation, modularity decreases with increasing GA. Higher modularity, as observed in younger fetuses, is indicative of more segregated functional subnetworks. While modules derived for younger and older fetuses showed similarities in their topological organization (e.g., the right hemisphere temporal insular cortex module was similarly distributed in both groups; [fig_ref] Figure 2: Macro-scale functional brain network modules for younger [/fig_ref] , blue), differences were noted for several modules. For example, sensorimotor cortices [fig_ref] Figure 3: Specific modular changes with development in younger versus older fetuses [/fig_ref] , red nodes, red circles) appear more functionally connected to regions of the cerebellum and inferior temporal cortices in older fetuses. Additionally, prefrontal areas [fig_ref] Figure 3: Specific modular changes with development in younger versus older fetuses [/fig_ref] , green nodes, green circles) appear more functionally connected to regions of the posterior parietal cortex in older fetuses. Furthermore, a ventral frontal temporal cortex module [fig_ref] Figure 3: Specific modular changes with development in younger versus older fetuses [/fig_ref] , teal nodes, teal circles) was present in both groups, but this module appeared to became more left lateralized and functionally integrated with frontal and inferior parietal regions in older fetuses. Only in older fetuses did this module encompass areas that will develop into Broca's and Wernicke's Areas, critical for speech comprehension and production. Finally, the occipital module [fig_ref] Figure 3: Specific modular changes with development in younger versus older fetuses [/fig_ref] , magenta nodes, magenta circles) appeared to better encompass the boundaries of primary and secondary visual cortex in older compared to younger fetuses. We also discovered a left, lateral parietal and premotor cortical module [fig_ref] Figure 3: Specific modular changes with development in younger versus older fetuses [/fig_ref] , yellow nodes) that was more prominent only in younger fetuses.
## Strength and length of functional brain connections
The strength of functional connectivity was positively related to distance between long-range (top 5%) brain areas in older but not younger fetuses. In older fetuses, the longest of the long connections (Euclidean distance .73.5 mm) were most strongly correlated (p = 0.004; mean strength (r) of long connections = 0.14). This did not appear to be a motion related artifact [bib_ref] Spurious but systematic correlations in functional connectivity MRI networks arise from subject..., Power [/bib_ref] [bib_ref] Heterogeneous impact of motion on fundamental patterns of developmental changes in functional..., Satterthwaite [/bib_ref] , because this comparison remained significant even when controlling for motion (r = 0.40, p = 0.02). No relationship was observed within the shortest connections (bottom 5%) for the older or younger fetal groups. Additionally, groups did not differ from one another in average strength of connectivity when assessing all possible connections. We did, however, observe a tighter range of pairwise correlation values with age, r = 0.35 p = 0.05, such that older fetuses showed correlation values (r) that were predominantly between 20.1 and 0.5 and younger fetuses showed a wider range of pairwise correlation values, see [fig_ref] Figure 4: Range and frequency of functional correlations [/fig_ref].
## Negative connectivity of the posterior cingulate cortex (pcc)
Intermodular negative strength Z-score for the PCC was significantly positively related to age (robust regression t = 3.5 p,0.002). That is, the fetal PCC module demonstrated more negative connection to other brain modules with advancing [fig_ref] Figure 5: Negative connectivity in the posterior cingulate cortex [/fig_ref]. This was the only region with this significant effect. Based on the finding that more negative intermodular functional connectivity of the PCC occurs with age, we performed an exploratory search for functional connections from the PCC that became more negative with age. One region that was in a different module than the PCC consistently across participants, the right lateral posterior cerebellum, also demonstrated a significant negative correlation of age and functional connectivity strength with the PCC (r = 20.35, p = 0.04). Other significant age correlations with regional graph theory measures (robust regression, p,0.002) included negative strength (or the sum of all negative connections to a given node): left middle peri-rolandic parietal lobe (t = 23.42) and left dorsal posterior cerebellum (t = 24.81); negative participation coefficient: right inferior occipital lobe (t = 3.41); positive within-module strength z-score: left parietal lobe (t = 3.78); and positive between-module strength z-score: left lateral peri-rolandic parietal lobe (t = 23.64) and right inferior occipital lobe (t = 3.66).
# Discussion
This study demonstrates the utility of fetal fMRI for discovering principles of neural system organization at the beginning of human life. Our results indicate the fetal brain is organized with modular structure, wherein connections are much stronger within than between modules. This is in agreement with observations in adults [bib_ref] Uncovering intrinsic modular organization of spontaneous brain activity in humans, He [/bib_ref] , and suggests modularity is an early emergent characteristic of the developing brain. We also found that with advancing gestational age intermodule connection strength and negative connectivity between the PCC and other brain regions increase. This study applied graph theoretical techniques, which are favorable for evaluation of fetal brain functional organization where a priori knowledge is limited.
Reduced intermodular connection strength and high modularity in younger fetuses suggest that in early fetal life functional systems are independent, and only with time do these begin to collaborate more fully as members of a whole brain system. Prior observations in late childhood, adolescence, and adulthood have provided mixed evidence about age-related independence of brain modules. Early research demonstrated that brain modules become increasingly independent and separable with advancing age [bib_ref] Functional brain networks develop from a ''local to distributed'' organization, Fair [/bib_ref] [bib_ref] Changes in the interaction of resting-state neural networks from adolescence to adulthood, Stevens [/bib_ref]. However, recent fcMRI research in large (N.100), motion-corrected samples describe linear decreases in modularity with age in participants ranging from ages 7 to 85 years [bib_ref] Topological organization of the human brain functional connectome across the lifespan, Cao [/bib_ref]. The apparent contradiction in these results may relate to experimental approach differences. In particular, the latter performed analyses across a wide age range, which may favor identification of life-span related patterns of change. Evaluation of brain modules in tighter age ranges may be more sensitive to developmental transitions, and these may show transitions from childhood to young adulthood are characterized by increased brain modularity. Our research extends the age range covered in these prior works by many years to provide the first evidence that modularity decreases in the prenatal brain. It is possible that development of functional circuitry in the antenatal brain proceeds in an inverted-U [bib_ref] The relation of strength of stimulus to rapidity of habit-formation, Yerkes [/bib_ref]. During pre-and post-natal life functional modules may develop, become integrated (possibly over integrated) then with development these may be pruned, or trimmed back, and functional [fig_ref] Figure 2: Macro-scale functional brain network modules for younger [/fig_ref] are depicted for the right and left lateral brain perspective and overlaid on a semi-transparent anatomical reference scan. Modules that show significant reconfiguration in older fetuses are highlighted. In older fetuses, sensorimotor cortices (red nodes, red circles) were more functionally connected to regions of the cerebellum and inferior temporal cortices. Prefrontal areas (green nodes, green circles) were more functionally connected to regions of the posterior parietal cortex in older fetuses. A ventral frontal temporal cortex module (teal nodes, teal circles) was present in both groups, but this module became more left lateralized and functionally integrated with inferior frontal and parietal regions in older fetuses. The occipital module (magenta nodes, magenta circles) expanded superiorly to encompass primary and secondary visual cortices in older fetuses. doi:10.1371/journal.pone.0094423.g003 substructures may become, again, more separable. The major difference apparent on either side of this proposed U is that the modules observed here in early fetal life are spatially restricted (i.e., dense local connectivity), whereas the modules observed in the shift to young adulthood span long distances in the brain, facilitating communication between widely dispersed regions [bib_ref] Development of distinct control networks through segregation and integration, Fair [/bib_ref] [bib_ref] Functional brain networks develop from a ''local to distributed'' organization, Fair [/bib_ref] [bib_ref] Restingstate fMRI can reliably map neural networks in children, Thomason [/bib_ref].
Here, we observe that modules obtained from graph analysis of the human fetal brain comprise areas that will later support vision, movement, language, and data integration (e.g., multimodal, association cortices). For example, we identified a module that spans somatosensory and motor cortices, resembling a network obtained by clustering analysis in a large (n = 1000) adult functional connectivity dataset [bib_ref] The organization of the human cerebral cortex estimated by intrinsic functional connectivity, Yeo [/bib_ref]. It appears that these systems become integrated during fetal life, and remain collaborative throughout the life span. Further, we noted a module that included regions of the default mode network (DMN) as defined in the extant neuroimaging literature [bib_ref] A default mode of brain function, Raichle [/bib_ref]. Discovering coordinated DMN function in utero is consistent with studies in term-and preterm-born infants indicating that a 'proto-default-mode network' is established in early life [bib_ref] Spontaneous brain activity in the newborn brain during natural sleep-an fMRI study..., Fransson [/bib_ref] [bib_ref] Restingstate networks in the infant brain, Fransson [/bib_ref]. We suggest that modules observed in fetuses may constitute developing functional systems, and acknowledge further work is needed to track the emergence of the default mode and other functional networks.
While similar brain networks (i.e., modules) were identified in older and younger fetuses, their spatial distributions differed. For instance, a module that largely corresponded to left lateralized language regions appeared to encompass Broca's and Wernicke's Areas in older but not younger fetuses. In addition, the cerebellum was included in a module that contained sensorimotor regions in older but not younger fetuses. This is consistent with fcMRI studies of adults that demonstrate intrinsic functional connections between motor and cerebellar regions [bib_ref] Functional topography of the cerebellum for motor and cognitive tasks: an fMRI..., Stoodley [/bib_ref] , reflecting underlying anatomical connectivity of these regions via thalamic pathways. The present work extends these observations by providing provisional maps of evolving neural connectivity systems present in fetal life.
Two observations in these data converge in support of a central role for the PCC in the development of functional brain networks in fetal life. First, we observed increased negative FC between the PCC and other brain regions with advancing gestational age [fig_ref] Figure 5: Negative connectivity in the posterior cingulate cortex [/fig_ref]. Second, we found that PCC/superior parietal regions [fig_ref] Figure 3: Specific modular changes with development in younger versus older fetuses [/fig_ref] , green) were functionally connected to regions of the prefrontal cortex in older fetuses only. Previous studies of both functional and structural networks have demonstrated the PCC is a major cortical connectivity hub [bib_ref] The precuneus/posterior cingulate cortex plays a pivotal role in the default mode..., Fransson [/bib_ref] [bib_ref] Mapping the structural core of human cerebral cortex, Hagmann [/bib_ref] [bib_ref] Precuneus shares intrinsic functional architecture in humans and monkeys, Margulies [/bib_ref] , even in infancy [bib_ref] Evidence on the emergence of the brain's default network from 2-week-old to..., Gao [/bib_ref]. The PCC is also a primary hub of the DMN [bib_ref] Functional-anatomic fractionation of the brain's default network, Andrews-Hanna [/bib_ref]. This network, comprised of PCC, medial prefrontal, and lateral parietal regions, has an anticorrelated relationship with the dorsal attention network [bib_ref] The human brain is intrinsically organized into dynamic, anticorrelated functional networks, Fox [/bib_ref] [bib_ref] Spontaneous low-frequency BOLD signal fluctuations: an fMRI investigation of the resting-state default..., Fransson [/bib_ref] [bib_ref] Default-mode function and task-induced deactivation have overlapping brain substrates in children, Thomason [/bib_ref] , and this dynamic relationship is compromised in disease [bib_ref] Default mode network activity and connectivity in psychopathology, Whitfield-Gabrieli [/bib_ref] , and mediates adaptive cognitive function [bib_ref] Functional connectivity between task-positive and task-negative brain areas and its relation to..., Hampson [/bib_ref] [bib_ref] Functional maturation of the executive system during adolescence, Satterthwaite [/bib_ref]. We present evidence that the negative opposition of the PCC to other brain networks begins to be established in human fetal life. Early and emergent anti-correlated function between the PCC and other brain regions suggests the PCC may serve a foundational role in the establishment of functional neural networks in the human fetal brain.
Methodological considerations of the current study warrant discussion. A chief consideration is that neurovascular coupling and cerebrovascular reactivity are not well understood in the human fetal brain. The BOLD fMRI connectivity approach is reliant upon reverse inference about the development of neural networks from patterns depicted by blood oxygen signals. Uncertainty about neural mechanisms that give rise to fMRI correlations is not unique to fetal imaging (see review by [bib_ref] Ongoing physiological processes in the cerebral cortex, Leopold [/bib_ref] , but immaturity of the fetal brain presents an added complexity. Available evidence from the adult brain indicates that functional connectivity is most highly correlated with neural local field potentials and gamma range activity [bib_ref] Ongoing physiological processes in the cerebral cortex, Leopold [/bib_ref] [bib_ref] Global and local fMRI signals driven by neurons defined optogenetically by type..., Lee [/bib_ref]. Considerations in the current study are that physiological properties underlying BOLD hemodynamic signals in our early and late fetuses may differ, and that we lack knowledge about how fetal BOLD hemodynamics in utero compare to postnatal hemodynamics. Despite evidence that BOLD fMRI hemodynamic responses are reliably identified in infants born prematurely [bib_ref] Development of BOLD signal hemodynamic responses in the human brain, Arichi [/bib_ref] , rodent studies show positive BOLD response is not detected in the rat brain until postnatal day eleven [bib_ref] Development of hemodynamic responses and functional connectivity in rat somatosensory cortex, Colonnese [/bib_ref] , which equates to approximately 28 weeks in human gestation. However, observations about the directionality of BOLD activity do not have direct bearing on conclusions drawn from the current data, as BOLD activity must not be confused with BOLD connectivity. It is possible that BOLD connectivity analysis of the present study is more robust to developmental changes in hemodynamic response properties than would a task based fMRI activation map, by comparison. We observed a trend for functional correlations to become more positive (shifting from more negative) and to have a more narrow range in distribution with increasing fetal age [fig_ref] Figure 4: Range and frequency of functional correlations [/fig_ref]. It is possible that these features depict important properties within development of neurovascular reactivity in human fetuses that are presently not known. With increased use of this methodology in human fetuses there will certainly be a need for increased study of the neurovascular elements of the obtained measures.
Another methodological consideration for the current work is the significant problem of fetal movement. Conventional approaches for motion correction cannot adequately address the magnitude and conditions of movement encountered in fetal fMRI. For example, because movements of the fetus are independent of the mother, but both are included in the acquired volume, standard tools for plotting, thresholding, or correcting movement are rendered ineffective. We have taken an approach in this and our prior work [bib_ref] Cross-hemispheric functional connectivity in the human fetal brain, Thomason [/bib_ref] that requires manual extraction of the fetal brain and realignment of volumes to the first volume of each protracted period of minimal movement, for which standard motion correction procedures are adequate. However, because younger fetuses move more than older fetuses, this approach resulted in significantly more analyzed frames in older fetuses. It may be useful for future studies to exclude even greater quantities of data in older fetuses so to constrain groups to having no difference in total number of volumes analyzed. We report loss of 39% of fMRI volumes to movement in this fetal sample. While this is extreme, (1) longer scans were collected in anticipation of this effect, leaving us with sufficient data of fair quality, and (2) this degree of data loss is similar to studies of adults that used stringent movement thresholds (35 to 39% [bib_ref] Spurious but systematic correlations in functional connectivity MRI networks arise from subject..., Power [/bib_ref]. As fetal fMRI is in nascent stages, considerable research remains to develop best practices for addressing the magnitude of movement and unique challenges inherent in fetal fcMRI methodology. While these methodological caveats make fetal FC more challenging to interpret, fcMRI remains a powerful new technique for non-invasively detecting neural connections in utero.
This study summarizes observations about development of brain networks prior to birth in healthy human fetuses. We found that as human fetuses advance in gestational age, intermodule connection strength increases, modularity decreases, modules begin to overlap with known functional systems, and function in the PCC becomes more negatively correlated with other brain areas. Understanding brain development in fetal life has relevance for both human neuroscience and for understanding the brain basis of common neurological problems that may not be observable using other imaging approaches. Neuroimaging research shows, for example, that brain maturation differs between the sexes [bib_ref] Sex differences in the structural connectome of the human brain, Ingalhalikar [/bib_ref] , and that infants born prematurely have altered neuroconnectivity [bib_ref] Brain injury in premature infants: a complex amalgam of destructive and developmental..., Volpe [/bib_ref] , but we have no knowledge about whether these differences emerge prenatally. There is a need to further develop fetal connectivity fMRI methodology and to apply this technique to the study of human fetal health and disease. Establishment of a new field of fetal fcMRI will provide an unprecedented means for evaluating brain development at the beginning of life, and will enable a platform for discovering the fetal origins or antecedents of neural injury or disease. thank G. H. Baldwin, B. Rihan, H. Nguyen, M. Nye, T. Lozon, and K. Martin, for assistance in data management and analyses. We thank L. Nikita, and J. Bieda for assistance with participant recruitment and communication. We thank the pregnant mothers and their families who participated in this research project for their invaluable contributions to our work.
# Author contributions
[fig] Figure 2: Macro-scale functional brain network modules for younger ( = 27.6 weeks; n = 17) and older ( = 34.4 weeks; n = 16) fetuses. Nodes are represented as colored spheres and edges are represented as the connections between nodes. Common colors are assigned to maximally similar modules in the two groups, but color assignments are arbitrary. doi:10.1371/journal.pone.0094423.g002 GA, as depicted by the positive regression line in [/fig]
[fig] Figure 3: Specific modular changes with development in younger versus older fetuses. Graph visualizations provided in [/fig]
[fig] Figure 4: Range and frequency of functional correlations (r) in younger and older fetuses. A bar plot of younger fetuses' correlation values subtracted from older fetuses' correlation values is shown. The majority of correlation values measured in older fetuses occurred between -.1 and.5. In contrast, a wider range of correlation values were measured in younger fetuses. Differences in the full range of correlation values between young and old groups showed a trend, r = 20.32, p = 0.07. doi:10.1371/journal.pone.0094423.g004 [/fig]
[fig] Figure 5: Negative connectivity in the posterior cingulate cortex (PCC). With advancing gestational age, intramodular anticorrelations of the PCC (in yellow, A) significantly increase (B), p,.05, FDR corrected. The PCC thus becomes more anticorrelated with other brain modules as fetuses mature. doi:10.1371/journal.pone.0094423.g005 [/fig]
[table] Table 1: Summary of Participant and Data Characteristics. [/table]
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Performance Optimization of an Air-Standard Irreversible Dual-Atkinson Cycle Engine Based on the Ecological Coefficient of Performance Criterion
This paper presents an ecological performance analysis and optimization for an air-standard irreversible Dual-Atkinson cycle (DAC) based on the ecological coefficient of performance (ECOP) criterion which includes internal irreversibilities, heat leak, and finite-rate of heat transfer. A comprehensive numerical analysis has been realized so as to investigate the global and optimal performances of the cycle. The results obtained based on the ECOP criterion are compared with a different ecological function which is named as the ecologic objective-function and with the maximum power output conditions. The results have been attained introducing the compression ratio, cut-off ratio, pressure ratio, Atkinson cycle ratio, source temperature ratio, and internal irreversibility parameter. The change of cycle performance with respect to these parameters is investigated and graphically presented.
# Introduction
In the recent years, the studies related to engine research focused on reducing pollutant emissions, particularly NO , released from internal combustion engines owing to environmental regulations and restrictions. The application of the Atkinson and Miller cycles to the internal combustion engines (ICE) may reduce NO emissions with little cost of power [bib_ref] Comparison of diesel engine and Miller cycled diesel engine by using two..., Gonca [/bib_ref] [bib_ref] Comparison of steam injected diesel engine and miller cycled diesel engine by..., Gonca [/bib_ref] [bib_ref] A study on late intake valve closing miller cycled diesel engine, Gonca [/bib_ref] [bib_ref] Performance maps for an airstandard irreversible Dual-Miller cycle (DMC) with late inlet..., Gonca [/bib_ref] [bib_ref] Application of the Miller cycle to reduce NOx emissions from petrol engines, Wang [/bib_ref] [bib_ref] An analytic study of applying Miller cycle to reduce NO emission from..., Wang [/bib_ref] [bib_ref] A comparison of Miller and Otto cycle natural gas engines for small..., Mikalsen [/bib_ref] [bib_ref] Efficiency improvement and NOx emission reduction potentials of two-stage turbocharged Miller cycle..., Kesgin [/bib_ref] [bib_ref] Experimental investigation of applying Miller cycle to reduce NO emission from diesel..., Wang [/bib_ref] [bib_ref] Potential of Atkinson cycle combined with EGR for pollutant control in a..., Benajes [/bib_ref]. In the literature, various optimization studies on the Miller and Atkinson cycles have been carried out. However, it should be noted that, at the same peak combustion temperatures and pressures, Atkinson cycle could become more efficient than the Miller cycle due to higher expansion ratio, as the amount of heat wasted with the exhaust gases may be reduced and transformed to power output by increasing expansion ratio. There are various studies on the Miller and Atkinson cycles. Gonca et al. [bib_ref] Comparison of diesel engine and Miller cycled diesel engine by using two..., Gonca [/bib_ref] [bib_ref] Comparison of steam injected diesel engine and miller cycled diesel engine by..., Gonca [/bib_ref] [bib_ref] A study on late intake valve closing miller cycled diesel engine, Gonca [/bib_ref] [bib_ref] Performance maps for an airstandard irreversible Dual-Miller cycle (DMC) with late inlet..., Gonca [/bib_ref] showed that the Miller cycle diesel engine is more advantageous than conventional diesel engine in terms of NO emissions and effective efficiency depending on numerical studies. Wang et al. experimentally [bib_ref] Application of the Miller cycle to reduce NOx emissions from petrol engines, Wang [/bib_ref] and analytically [bib_ref] An analytic study of applying Miller cycle to reduce NO emission from..., Wang [/bib_ref] implemented the Miller cycle into a petrol engine in order to decrease the NO emissions. Mikalsen et al. [bib_ref] A comparison of Miller and Otto cycle natural gas engines for small..., Mikalsen [/bib_ref] applied the Miller cycle into an Otto cycle natural gas engine and the SFC and power output of the engine decreased. Kesgin [bib_ref] Efficiency improvement and NOx emission reduction potentials of two-stage turbocharged Miller cycle..., Kesgin [/bib_ref] experimentally and theoretically applied the Miller cycle into a natural gas engine and the efficiency increased and NO emissions could be abated. Wang et al. [bib_ref] Experimental investigation of applying Miller cycle to reduce NO emission from diesel..., Wang [/bib_ref] experimentally applied the Miller cycle into a diesel engine and NO emissions decreased. Lin and Hou [bib_ref] Performance analysis of an air-standard Miller cycle with considerations of heat loss..., Lin [/bib_ref] expressed that the performance of Miller cycle is higher than that of Otto cycle at same peak temperature conditions. The influences of temperature-dependent specific heats of the working fluid on the performance characteristics were investigated for an air-standard reversible Miller cycle [bib_ref] Efficiency of a Miller engine, Al-Sarkhi [/bib_ref] and irreversible Miller cycle with different specific heat models [bib_ref] Performance evaluation of irreversible Miller engine under various specific heat models, Al-Sarkhi [/bib_ref] ; the total cycle volumes and pressure ratios of the Miller cycle were depicted with graphics at maximum power density conditions [bib_ref] Efficiency of miller engine at maximum power density, Al-Sarkhi [/bib_ref] by Al-Sarkhi et al., and Zhao and Chen [bib_ref] Performance analysis of an irreversible Miller heat engine and its optimum criteria, Zhao [/bib_ref] analyzed the performance of an air-standard irreversible Miller cycle by introducing the pressure ratios and considering the irreversibilities during the cycle processes. Wang et al. [bib_ref] Experimental investigation of applying Miller cycle to reduce NOx emission from diesel..., Wang [/bib_ref] experimentally applied the Miller cycle into a diesel 2
The Scientific World Journal engine. Wu et al. [bib_ref] Performance analysis and optimization of a supercharged Miller cycle Otto engine, Wu [/bib_ref] theoretically applied the Miller cycle into a supercharged Otto engine. Ebrahimi [bib_ref] Thermodynamic modeling of performance of a Miller cycle with engine speed and..., Ebrahimi [/bib_ref] [bib_ref] Performance analysis of an irreversible Miller cycle with considerations of relative air-fuel..., Ebrahimi [/bib_ref] conducted thermodynamical analyses for reversible Miller cycle with considerations of engine speed and variable specific heat ratio of working fluid [bib_ref] Thermodynamic modeling of performance of a Miller cycle with engine speed and..., Ebrahimi [/bib_ref] and for irreversible Miller cycle with respect to the variation of relative air-fuel ratio and stroke length [bib_ref] Performance analysis of an irreversible Miller cycle with considerations of relative air-fuel..., Ebrahimi [/bib_ref]. Wang and Hou [bib_ref] Performance analysis and comparison of an Atkinson cycle coupled to variable temperature..., Wang [/bib_ref] conducted a performance analysis for an Atkinson cycle coupled with variable temperature heat reservoirs under maximum power (MP) and maximum power density (MPD) conditions. Chen et al. [bib_ref] Efficiency of an Atkinson engine at maximum power density, Chen [/bib_ref] optimized the air-standard Atkinson cycle based on the MPD criterion. Al-Sarkhi et al. [bib_ref] Efficiency of atkinson engine at maximum power density using temperature dependent specific..., Al-Sarkhi [/bib_ref] expanded the study in [bib_ref] Efficiency of an Atkinson engine at maximum power density, Chen [/bib_ref] using temperature-dependent specific heat model and it was emphasized that this model has substantial influence on the performance of the Atkinson cycle. Ust [bib_ref] A comparative performance analysis and optimization of the irreversible atkinson cycle under..., Ust [/bib_ref] conducted a performance analysis and optimization for the irreversible Atkinson cycle by considering the internal irreversibilities originating from the adiabatic compression and expansion processes in order to define the optimum performance and design parameters of the cycle. Zhao and Chen [bib_ref] Performance analysis and parametric optimum criteria of an irreversible Atkinson heat-engine, Zhao [/bib_ref] analyzed an irreversible Atkinson cycle by taking account of irreversibilities originating from the adiabatic processes, finitetime processes, and heat transfer through the cylinder wall. Gahruei et al. [bib_ref] Mathematical modeling and comparison of air standard Dual and Dual-Atkinson cycles with..., Gahruei [/bib_ref] compared the performances of the classical Dual and Dual-Atkinson cycles based on finite-time thermodynamics by considering variable specific heats of the working fluid, heat transfer, and friction losses. Ge et al. [bib_ref] Performance of endoreversible Atkinson cycle, Ge [/bib_ref] carried out a performance optimization for an endoreversible Atkinson heat engine. Ge et al. [bib_ref] Performance of an Atkinson cycle with heat transfer, friction and variable specific-heats..., Ge [/bib_ref] investigated the influences of variable specific heats of the working fluid on the performance of Atkinson cycle. Zhao et al. [bib_ref] Design and optimization of an Atkinson cycle engine with the Artificial Neural..., Zhao [/bib_ref] performed an experimental and numerical study to design and optimize an Atkinson cycle engine by using Artificial Neural Network Method. Zhao and Xu [bib_ref] Fuel economy optimization of an Atkinson cycle engine using genetic algorithm, Zhao [/bib_ref] improved the fuel economy of an Atkinson cycle engine up to 7.67% by using the Genetic Algorithm. Ebrahimi [bib_ref] Effects of mean piston speed, equivalence ratio and cylinder wall temperature on..., Ebrahimi [/bib_ref] carried out a performance optimization of an Atkinson cycle heat engine by taking into account the impacts of the cylinder wall temperature, mean piston velocity, and equivalence ratio. Lin and Hou [bib_ref] Influence of heat loss on the performance of an air-standard Atkinson cycle, Lin [/bib_ref] examined the impacts of variable specific heats of the working fluid, friction, and losses, as a percentage of fuel's energy, on the performance of an air-standard Atkinson cycle. Hou [bib_ref] Comparison of performances of air standard Atkinson and Otto cycles with heat..., Hou [/bib_ref] compared the performances of the air-standard Otto and Atkinson cycles by taking into consideration the heat transfer impacts.
The studies related to performance optimization and thermodynamical analyses of engine cycles were realized with various methods and objective functions. One of the objective functions commonly used is the ecological objective-function proposed by Angulo-Brown [bib_ref] An ecological optimization criterion for finite-time heat engines, Angulo-Brown [/bib_ref]. This function is determined as the power output minus the loss rate of availability. In recent years, a new thermoecological objective-function has been developed by Ust et al. [bib_ref] Ecological performance analysis and optimization of power generation systems, Ust [/bib_ref] [bib_ref] Performance analysis and optimization of an irreversible dual-cycle based on an ecological..., Ust [/bib_ref]. This objective-function is called the ecological coefficient of performance (ECOP), which is stated as the proportion of the power output to the loss rate of availability. It was asserted that the ECOP criterion is more understandable compared to ecological objectivefunction [bib_ref] Performance analysis and optimization of an irreversible dual-cycle based on an ecological..., Ust [/bib_ref] , as the minimum entropy is formed at the maximum ECOP conditions. Various studies have been performed by applying the ECOP criterion to the heat engines [bib_ref] Ecological coefficient of performance (ECOP) optimization for generalized irreversible Carnot heat engines, Üst [/bib_ref] [bib_ref] Performance analysis of an irreversible Brayton heat engine based on ecological coefficient..., Ust [/bib_ref] [bib_ref] Ecological coefficient of performance (ECOP) optimization for an irreversible brayton heat engine..., Ust [/bib_ref] [bib_ref] Ecological coefficient of performance analysis and optimization of an irreversible regenerative-Brayton heat..., Ust [/bib_ref] [bib_ref] The effects of intercooling and regeneration on the thermo-ecological performance analysis of..., Sogut [/bib_ref] [bib_ref] Performance optimization of irreversible refrigerators based on a new thermo-ecological criterion, Ust [/bib_ref] [bib_ref] Performance analysis and optimization of irreversible air refrigeration cycles based on ecological..., Ust [/bib_ref]. Ust et al. [bib_ref] Performance analysis and optimization of an irreversible dual-cycle based on an ecological..., Ust [/bib_ref] carried out an ecological performance analysis for an irreversible dual cycle based on the ECOP criterion considering finiterate of heat transfer, heat leak, and internal irreversibilities. Ust et al. [bib_ref] Ecological coefficient of performance (ECOP) optimization for generalized irreversible Carnot heat engines, Üst [/bib_ref] [bib_ref] Performance analysis of an irreversible Brayton heat engine based on ecological coefficient..., Ust [/bib_ref] [bib_ref] Ecological coefficient of performance (ECOP) optimization for an irreversible brayton heat engine..., Ust [/bib_ref] [bib_ref] Ecological coefficient of performance analysis and optimization of an irreversible regenerative-Brayton heat..., Ust [/bib_ref] carried out performance analyses and optimizations for irreversible Carnot heat engine [bib_ref] Ecological coefficient of performance (ECOP) optimization for generalized irreversible Carnot heat engines, Üst [/bib_ref] and Brayton heat engine [bib_ref] Performance analysis of an irreversible Brayton heat engine based on ecological coefficient..., Ust [/bib_ref] [bib_ref] Ecological coefficient of performance (ECOP) optimization for an irreversible brayton heat engine..., Ust [/bib_ref] [bib_ref] Ecological coefficient of performance analysis and optimization of an irreversible regenerative-Brayton heat..., Ust [/bib_ref] considering losses owing to heat leak, heat transfer, and internal irreversibilities, based on ecological coefficient of performance (ECOP) function. Sogut et al. [bib_ref] The effects of intercooling and regeneration on the thermo-ecological performance analysis of..., Sogut [/bib_ref] investigated the influences of intercooling and regeneration on the thermoecological performance analysis of an irreversible-closed Brayton heat engine with variable temperature thermal reservoirs. Ust and Sahin [bib_ref] Performance optimization of irreversible refrigerators based on a new thermo-ecological criterion, Ust [/bib_ref] carried out a performance optimization for irreversible refrigerators based on the ECOP criterion. Ust [bib_ref] Performance analysis and optimization of irreversible air refrigeration cycles based on ecological..., Ust [/bib_ref] carried out a performance analysis based on ECOP criterion for irreversible air refrigeration cycles considering irreversibilities because of finite-rate heat transfer, heat leakage, and internal dissipations. This study presents a thermoecological performance analysis based on the ECOP, the ecological objective-function (Ė), and the maximum power output conditions for an irreversible DAC engine. The influences of the engine design parameters on the engine performance were examined. The general and optimal design parameters which give the maximum ECOP, the maximumĖ, and the maximum power output have been computationally determined. In the literature, there is no such study which applies theĖ and ECOP function on an irreversible DAC engine. Therefore, this study could be used as a guideline by real engine designers to obtain maximum ecological performance for DAC engines.
## Theoretical analysis of dac
-and -diagrams of the irreversible air-standard DAC (1-2-3-4-5-1) coupled with constant hot and cold temperature heat-reservoirs are depicted in [fig_ref] Figure 1: -schematic diagram for DAC [/fig_ref]. It is clear that the The Scientific World Journal 3 process 1-2 is an irreversible compression and internal irreversibilities were taken into account, whilst the process 1-2 s is an isentropic compression. The heat input is provided in the processes 2-3 (at constant volume) and 3-4 (at constant pressure). The process 4-5 is an irreversible expansion and internal irreversibilities were considered, while the process 4-5 s is an isentropic expansion. The heat rejection occurs in the process 5-1 (at constant pressure) and the cycle is completed.
In DAC cycle,̇1 anḋ2 are the heat transfer rates (time-dependent) from the hot resource at temperature to the working fluid (ideal air) in the processes 2-3 and 3-4;
is the heat transfer rate from the working fluid to the coldreservoir at temperature in the process 5-1.̇1,̇2, anḋ are expressed by expanding Ust et al. 's study [bib_ref] Performance analysis and optimization of an irreversible dual-cycle based on an ecological..., Ust [/bib_ref] as follows:̇1
[formula] = 1 1 ( − 2 ) − ( − 3 ) ln (( − 2 ) / ( − 3 )) =̇1 ( − 2 ) =̇( 3 − 2 ) ,(1)2 = 2 2 ( − 3 ) − ( − 4 ) ln (( − 3 ) / ( − 4 )) =̇2 (1 − 1 ) ( − 2 ) =̇( 4 − 3 ) ,(2)= ( 5 − ) − ( 1 − ) ln (( 5 − ) / ( 1 − )) =̇(1 − ) ( 5 − ) =̇( 5 − 1 ) ,(3) [/formula]
=̇,
where 1 1 , 2 2 , and are the conductance of the hot-reservoir at constant volume and at constant pressure and the conductance of cold-reservoir heat exchanger, respectively.̇is the capacity of the working fluid and is the isentropic exponent that is stated as the ratio of the specific heat at constant pressure to the specific heat at constant volume ( / ) of working fluid. 1 , 2 , and are the effectiveness of the hot-and cold-reservoirs of the heat exchanger which are expressed as below:
[formula] 1 = 1 − exp (− 1 ) ; 2 = 1 − exp (− 2 ) ; = 1 − exp (− ) ,(5) [/formula]
where 1 , 2 , and are the number of heat transfer units for hot-side and cold-side based on the minimum thermal capacity rates and they could be given as below:
[formula] 1 = 1 1 ; 2 = 2 2 ; =̇.(6) [/formula]
The heat leakage rate,̇, from the hot source at temperature to the cold source at temperature could be written as follows:̇=̇(
[formula] − ) =̇( − ) ,(7) [/formula]
wherėis the internal conductance of the DAC and is the percentage of the internal conductance with respect to the thermal capacity rate of the working fluid. The total heat rate, , transferred from hot-side is written aṡ
[formula] =̇{[ 1 + 2 (1 − 1 )] ( − 2 ) + ( − )} ,(8) [/formula]
and the total heat rate,̇, transferred to the cold-side is expressed aṡ
[formula] =̇{[ 1 + 2 (1 − 1 )] ( 5 − ) + ( − )} .(9) [/formula]
We can obtain below equations using (1)- (3):
[formula] 3 = 1 + (1 − 1 ) 2 , 4 = [ 1 + 2 (1 − 1 )] + (1 − 1 ) (1 − 2 ) 2 ,(10)1 = + (1 − ) 5 .(11) [/formula]
The net power output may be stated by using the first law of thermodynamics:
[formula] = (̇1 +̇2) −̇, =̇{[ 1 + 2 (1 − 1 )] ( − 2 ) − (1 − ) ( 5 − )} .(12) [/formula]
From (13),
[formula] 5 = + [ 1 + 2 (1 − 1 )] ( − 2 ) (1 − ) −( 1 − ) .(13) [/formula]
Substituting (13) into (11) we obtain the equation as below:
[formula] 1 = + (1 − ) × [ + [ 1 + 2 (1 − 1 )] ( − 2 ) (1 − ) −( 1 − ) ] .(14) [/formula]
The isentropic efficiencies of the compression and expansion processes are given as below [bib_ref] Performance maps for an airstandard irreversible Dual-Miller cycle (DMC) with late inlet..., Gonca [/bib_ref] :
[formula] = 2 − 1 2 − 1 ; = 4 − 5 4 − 5 .(15) [/formula]
4
The Scientific World Journal
The following equation is found based on the second law of thermodynamics:
[formula] 1 1− 3 4 = 2 5 .(16) [/formula]
and define the irreversibilities of the adiabatic processes. By using thermodynamic relations between the state points 1-5 and (15)-(16), the following equations are acquired:
[formula] ( 2 1 ) = + ( −1 − 1) ,( 5 4) = {1 − [1 − ( ) −1 ]} ,(17) [/formula]
where the compression ratio ( ) is given as
[formula] = V 1 V 2 .(18) [/formula]
Also, the following equation is given based on the second law of thermodynamics [bib_ref] Performance analysis and optimization of an irreversible dual-cycle based on an ecological..., Ust [/bib_ref] :
[formula] Δ 42 − Δ 51 < 0.(19) [/formula]
The inequality in (21) could be reordered as follows:
[formula] Δ Δ 42 − Δ 51 = 0 with Δ ≥ 1,(20) [/formula]
where Δ is internal irreversibility parameter and it is defined as
[formula] Δ = ( 5 − 1 ) ( 4 − 3 ) + ( 3 − 2 ) [/formula]
.
Consequently, the following equation is written as follows:
[formula] 1 Δ (1− ) 3 Δ 4 = Δ 2 5 .(22) [/formula]
In the study, dimensionless engine design parameters are the pressure ratio ( ), cut-off ratio ( ), and source temperatureratio ( ) and they can be given, respectively, as
[formula] = 3 2 = 3 2 ; = V 4 V 3 = 4 3 ; = .(23) [/formula]
The Atkinson cycle ratio ( ) is derived as Miller cycle ratio [bib_ref] Application of the Miller cycle to reduce NOx emissions from petrol engines, Wang [/bib_ref] and the Atkinson cycle ratio and stroke ratio may be expressed as follows:
[formula] = V 5 V 1 = 5 1 ; = V 5 V 2 = .(24) [/formula]
The entropy generation rate of DAC could be written aṡ
[formula] =̇−̇.(25) [/formula]
The ecological objective-function [bib_ref] Performance analysis and optimization of an irreversible dual-cycle based on an ecological..., Ust [/bib_ref] is given as below:
[formula] =̇− 0̇.(26) [/formula]
The ECOP criterion is attained as the proportion of the power output to the loss rate of availability as follows [bib_ref] Ecological performance analysis and optimization of power generation systems, Ust [/bib_ref] [bib_ref] Performance analysis and optimization of an irreversible dual-cycle based on an ecological..., Ust [/bib_ref] :
[formula] ECOP =0̇.(27) [/formula]
The thermal efficiency may be stated as follows:
=̇.
The figures are plotted in the next chapter by using the equations given above.
# Results and discussion
In this section, comprehensive computations are performed evaluating compression ratio ( ) in order to compare the consequences of DAC depending on different performance parameters such as maximum ECOP, MEF, and MP conditions. The figures in the text are plotted using numerical results. In the calculations, the constants are taken as = 1.4 and = = 0.9 and the total number of heat transfer units is given as below:
[formula] = 1 + 2 + , 1 = 2 .(29) [/formula]
The It is clearly seen from these figures that the ECOP,Ė, anḋincrease as the source temperature ratio ( ) rises and the internal irreversibility parameter ( Δ ) decreases [bib_ref] Performance analysis and optimization of an irreversible dual-cycle based on an ecological..., Ust [/bib_ref]. The Atkinson cycle ratio ( ) increases as the ECOP andĖ for the samėvalues and the maximuṁabates with raising of . It is clearly seen from that the ECOP increases to a maximum value and then starts to decrease. Nevertheless,Ė continuously raises with the increase oḟ. The curves given for the ECOP have more parabolic characteristics although those ofĖ are linear.
It is seen from Figures 2(a) and 2(b) that the maximum ECOP (ECOP MAX ) is higher than the ECOP at maximumĖ (ECOP MEF ) and ECOP at the maximuṁ(ECOP MP ). The order for the ECOP values can be given as ECOP MP < ECOP MEF < ECOP MAX . However, the order is ECOP MP = ECOP MEF < ECOP MAX for a specified value.
The variations of the ECOP anḋwith respect to for different and Δ are demonstrated in , respectively. It is observed from the figures that the ECOP anḋ raise to a certain value and then begin to abate, while
The Scientific World Journal increases. It is obvious that the has optimal values which give the maximum ECOP anḋ. These optimum values raise while raises and Δ diminishes. The optimal values for the maximuṁare higher compared to those of the ECOP in all conditions. Normalized forms of the ECOP, dimensionless power output, and ecological function with respect to the dimensionless entropy generation rate (̇=̇/̇) are depicted in [fig_ref] Figure 4: Variations of the normalized ECOP,̇anḋwith respect to dimensionless entropy generation rate [/fig_ref].̇M P ,̇M EF , anḋ * stand for the dimensionless entropy generation rate at the maximum dimensionless power output (̇M AX ), at the maximum ecological function (̇M AX ), and at the maximum ECOP (ECOP MAX ), respectively. The order may be expressed aṡ * <̇M EF <̇M P . This result is similar to that of [bib_ref] Performance analysis and optimization of an irreversible dual-cycle based on an ecological..., Ust [/bib_ref]. [fig_ref] Figure 5: Comparisons of the optimum performances at ECOP MAX , MAX , anḋM... [/fig_ref] shows the comparison of the performance parameters of ,̇, anḋfor ECOP MAX ,̇M AX , anḋM AX conditions. It is understood from the figures that the ECOP MAX circumstances are more advantageous compared tȯM AX circumstances in point oḟand thermal efficiency ( ), however, contrarily, disadvantageous compared to that with regard tȯcircumstances.
We see from the figures that the ECOP MAX conditions for the entropy generation rate and thermal efficiency and the disadvantage for the dimensionless power output raise while Δ reduces. The results show that ECOP MAX circumstances are more advantageous oveṙM AX circumstances up to 35%, 4% and disadvantageous up to 33% in points oḟ, , anḋ, respectively; ECOP MAX circumstances are more advantageous oveṙM AX circumstances up to 58%, 22% and disadvantageous up to 29% in points oḟ, anḋ, respectively. It should be noted that the variation of Δ remarkably affectṡM AX circumstances, even though it has no notable influence oṅM AX circumstances. The substantial nondimensional engine design parameters are , , , and . The results of the analysis based on these parameters are illustrated in . It is clear that aṫM AX circumstances ( MP ) is smallest compared to those at ECOP MAX ( * ) anḋM AX ( MEF ) circumstances. Hence, the relation between the optimal compression ratios for different conditions may be expressed as * > MEF > MP . On the other hand, the relation between Atkinson cycle ratios ( ), pressure ratios ( ), and cut-off ratios ( ) is just the opposite as may be seen from . The order between these parameters could be stated as * < ,MEF < ,MP , * < MEF < MP , and * < MEF < MP . Also, it may be observed that the order is < < at same maximum conditions. demonstrates the variation of the ECOP with respect to the and for different Atkinson cycle ratios. The ECOP abates, while raises for the same and values. It may be also seen from the figure that the maximum is smaller than at the same values. [fig_ref] Figure 8: versus at ECOP MAX ,̇M AX , anḋM AX circumstances for different... [/fig_ref] illustrates the variation of values with respect to for different Δ values.
values at ECOP MAX ( * ) are smaller than those aṫM AX ( ,MEF ) anḋM AX ( ,MP ); this relationship is similar to those of and . The relation can be written as * < ,MEF < ,MP . As may be observed from the figure, values at the maximum conditions raise with increasing of . For specified , ,MEF reduces steadily, whilst Δ raises. Nevertheless, the variation of * and ,MP is unstable with the variation of Δ .
The impacts of Δ and on the variation of the optimum and in terms of ECOP MAX ,̇M AX , anḋM AX are illustrated in [fig_ref] Figure 1: -schematic diagram for DAC [/fig_ref] , respectively. It is seen from the figures that the optimum values of the engine design parameters increase as raises for a determined Δ value at the ECOP MAX ,̇M AX , anḋM AX conditions. One can see that reduces and raises with the increase of Δ for a specified value. However, increase rate of ,MEF and ,MP is higher compared to that of * . Therefore, the optimum The Scientific World Journal values of the engine design parameters change with respect to the determined objective-function, for a specified .
# Conclusion
A thermoecological performance analysis has been conducted so as to define the optimum engine operation and design parameters for the air-standard irreversible DAC cycle having a finite-rate of heat transfer, heat leakage and internal irreversibilities based on the ecological function, the maximum power output, and the ECOP criteria.
In this standpoint, the optimum pressure ratio ( ), cut-off ratio ( ), compression ratio ( ), and the Atkinson cycle ratio ( ) that maximize the ecological coefficient of performance (ECOP MAX ), ecological function (̇M AX ), and power output (̇M AX ) have been examined. And also a comparative study based on maximum values of these criteria has been realized for an irreversible Atkinson cycled engine model; the relations between the power output and ECOP and the Atkinson cycle ratio and ECOP for different and Δ values have been derived. The ECOP,Ė, anḋversus and figures for the DAC have been illustrated for and Δ values. It is noted that ECOP MAX circumstances have substantial vantage over thė MAX circumstances in point of entropy formation rate and little vantage in point of thermal efficiency: on the other hand, a slight disadvantage is observed in point of power output.
Comparisons at the maximum power output conditions (̇M AX ) show that ECOP MAX circumstances have significant vantages in terms of ecological viewpoints with little loss of power output. The optimum , , , and values at ECOP MAX ,̇M AX , anḋM AX conditions with respect to the variation of and Δ have been depicted so as to provide good guidelines for the description of the optimal design and operating conditions of real Atkinson cycle diesel engines.
## Nomenclature
:
Heattransferarea(m 2 ) :
Specific heat at constant pressure (kW/kg ⋅ K) :̇(kW/K) DAC: Dual-Atkinson cyclė E:
Ecological performance function ECOP: Ecological coefficient of performance Δ :
Internal irreversibility parameter :
Isentropic exponenṫ :
Massflowrate(kg/s) :
Total number of heat transfer units :
Pressure (kPa) :
Rate of heat transfer (kW) :
Compression ratio = V 1 /V 2 :
Atkinson cycle ratio, = V 6 /V 1 :
Entropy(kJ/K) :
T emperature(K) :
Overall heat transfer coefficient (kW/m 2 K) :
V olume(m 3 ) :
Poweroutput(kW).
## Greek letters
: Stroke ratio, = V 6 /V 2 : Pressure ratio, = 3 / 2 : Heat-exchanger effectiveness : Allocation ratio ( 1 + 2 )/ : Thermal efficiency : Isentropic efficiency of compression : Isentropic efficiency of expansion : Cut-off ratio, = 4 / 3 : Source temperature ratio = / .
## Subscripts
## : generation
: High-temperature heat-source : Low-temperature heat-source The Scientific World Journal 9 MAX: Maximum MEF: At the maximum ecological objectivefunction condition MP: At maximum power output condition 0:
Environment condition.
Superscripts * : At the maximum ECOP condition : Dimensionless.
## Conflict of interests
The authors declare that there is no conflict of interests regarding the publication of this paper.
[fig] Figure 1: -schematic diagram for DAC. [/fig]
[fig] Figure 2, Figure 3: Variations of the ECOP and ecological function with respect to nondimensional power output for (a) various ( Δ = 1.2); (b) for various Δ ( = 7); (c) for various ( Δ = 1.2, = 17) ( 0 = = 300 K, = ( Variations of the ECOP and dimensionless power output with respect to Atkinson cycle ratio for (a) various , ( Δ = 1.2); and (b) for various Δ ( = 7) ( 0 = = 300 K, = ( 1 + 2 )/ = 0.3, and = 0.02). [/fig]
[fig] Figure 4: Variations of the normalized ECOP,̇anḋwith respect to dimensionless entropy generation rate ( 0 = = 300 K, Δ = 1.2, = 7, = 0.3, and = 0.02). [/fig]
[fig] Figure 5: Comparisons of the optimum performances at ECOP MAX , MAX , anḋM AX conditions based on Δ for = 7 ( 0 = = 300 K, = 0.3, and = 0.02). [/fig]
[fig] Figure 6, Figure 7: Normalized ECOPĖ anḋversus (a) and (b) , , ( 0 = = 300 K, = 7, Δ = 1.2, = 0.3, and = 0.02). ECOP versus , for different Atkinson cycle ratios ( Δ = 1.2, = 17) ( 0 = = 300 K, = 0.3, and = 0.02). [/fig]
[fig] Figure 8: versus at ECOP MAX ,̇M AX , anḋM AX circumstances for different Δ values ( 0 = = 300 K, = 17 = 0.3, and = 0.02). [/fig]
[fig] Figure 9: Variation of the optimum at ECOP MAX circumstances based on the optimum , for various and Δ values ( 0 = = 300 K, = 0.3, and = 0.02). [/fig]
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Autonomic aging – A dataset to quantify changes of cardiovascular autonomic function during healthy aging
autonomic regulation of blood pressure and cardiac rhythm progressively declines with increasing age. Impaired cardiovascular control promotes a variety of age-related cardio-vascular conditions. this study aims to provide a database of high-resolution biological signals to describe the effect of healthy aging on cardiovascular regulation. Electrocardiogram and continuous non-invasive blood pressure signals were recorded simultaneously at rest in 1,121 healthy volunteers. With this database, we provide raw signals as well as basic demographic information such as gender and body mass index. to demonstrate validity of the acquired data, we present the well-known decline of heart rate variability with increasing age in this database.
# Background & summary
As the elderly population has a high prevalence of cardiovascular diseases, representing the globally leading cause of death, it is essential to detect bodily changes that adumbrate cardiovascular impairments. In addition, impairment of autonomic regulation of the cardiovascular system seems to accelerate the decline of cognitive performance and promotes the risk to develop age-related diseases.
It is well established that arterial and ventricular stiffening, reduction of myocardial contractility, or degeneration of organ innervation impair cardiovascular function in an aged population and promote sustained hypertension, thrombosis, and atherosclerosis [bib_ref] Cardiovascular physiology in the older adults, Dai [/bib_ref]. Recent scientific studies have suggested that an elevation of blood pressure and signs of thrombosis in midlife increase the risk to develop Alzheimer's disease or other dementias in later life [bib_ref] Cumulative blood pressure exposure during young adulthood and mobility and cognitive function..., Mahinrad [/bib_ref] [bib_ref] Cardiovascular health metrics from mid-To late-life and risk of dementia: A population-based..., Liang [/bib_ref].
A decreased sensitivity of vagal reflexes such as respiratory sinus arrhythmia leads to elevated heart rates and diminished heart rate variability in elderly participants [bib_ref] Age dependency of cardiovascular autonomic responses to head-up tilt in healthy subjects, Laitinen [/bib_ref] [bib_ref] Influence of Age on Linear and Nonlinear Measures of Autonomic Cardiovascular Modulation, Boettger [/bib_ref]. The feedback loop decelerating heart rate due to increasing blood pressure, i.e. baroreflex function, is progressively diminished with increasing age. Structural changes such as the loss of sinoatrial pacemaker cells and left ventricular hypertrophy as well as functional changes of receptors initiating autonomic reflexes (e.g. baroreceptors) contribute to an altered cardiovascular regulation related to aging [bib_ref] Effect of age and high blood pressure on baroreflex sensitivity in man, Gribbin [/bib_ref] [bib_ref] Assessment of the primary effect of aging on heart rate variability in..., Fukusaki [/bib_ref].
Lower levels of vagally modulated HRV have been associated with increased cardiovascular morbidity and mortality in the elderly 12 . Hillebrand et al. reported that healthy participants with diminished resting heart rate variability (HRV) have a 32-45% increased risk to suffer from a first cardiovascular event [bib_ref] Heart rate variability and first cardiovascular event in populations without known cardiovascular..., Hillebrand [/bib_ref]. Resting heart rate and HRV were demonstrated to predict cardiovascular disease as well as cardiac and overall mortality [bib_ref] Elevated resting heart rate, physical fitness and all-cause mortality: A 16-year follow-up..., Jensen [/bib_ref].
In this study, we enrolled healthy volunteers between 2010 to 2020. Participants were recruited from the local community as well as employees of the University Hospital. We recorded electrocardiograms and continuous noninvasive blood pressure at carefully controlled resting state conditions in our laboratory with a constant well-defined room temperature and humidity, a defined illumination level and without any environmental noise. Available data might be used to describe the influence of aging on cardiovascular autonomic regulation to better understand mechanisms of healthy aging in contrast to pathological changes. www.nature.com/scientificdata www.nature.com/scientificdata/ Methods participants. Resting state physiological recordings of 1,121 healthy volunteers were obtained. Exclusion criteria were any medical conditions, illegal drugs or medication potentially influencing cardiovascular function. Thorough physical examination, resting electrocardiography (ECG) and routine laboratory parameters (electrolytes, basic metabolic panel, and blood count) had to be without any pathological finding (results are not part of the database). Demographic information is depicted in .
Human subjects. The study was approved by the ethics committee of the Friedrich-Schiller-Universität Jena.
All research was performed in accordance with relevant guidelines and regulations. Informed written consent and consent for anonymous data sharing were obtained from all participants.
## Procedures. all measurements were recorded at the department of psychosomatic medicine and
Psychotherapy at Jena University Hospital.
EcG. An ECG (lead II) was recorded at 1000 Hz either by an MP150 (ECG100C, BIOPAC systems inc., Golata, CA, USA) or Task Force Monitor system (CNSystems Medizintechnik GmbH, Graz, AUT). Pre-gelled Ag/AgCl electrodes (BlueSensor VL, Ambu GmbH, Bad Nauheim, GER) were attached according to an Einthoven triangle [fig_ref] Figure 1: Cardiovascular recordings using system 1 -the Task Force Monitor [/fig_ref]. ECG and continuous blood pressure were digitalized synchronously by the analog-to-digital converter incorporated in the main system (MP150 or Task Force Monitor).
Blood pressure. Continuous blood pressure was recorded non-invasively using the vascular unloading technique [bib_ref] Continuous non-invasive blood pressure monitoring using concentrically interlocking control loops, Fortin [/bib_ref]. In short, a cuff around the finger is controlled to maintain constant pressure, while blood volume is recorded via photoplethysmography. With non-varying cuff pressure, the acquired blood volume is proportional to blood pressure in the arteries of the finger. The recorded signal is calibrated by an oscillometric measurement of the brachial blood pressure once during initialization of the system (measurement accuracy ± 3 mmHg). The Task Force Monitor is equipped with a module for continuous blood pressure measurement. The MP150 system digitizes the signal acquired by a separate monitor CNAP 500 (CNSystems Medizintechnik GmbH, Graz, AUT). The sampling frequency was 100 Hz for both systems.
Missing data. During some recordings, the system measured brachial blood pressure again for re-calibration.
This led to loss of continuous NIBP signal over a short time span. Missing data points have been set to NaN. When demographic data was missing, respective cells were filled with NaN.
parameters. To demonstrate plausibility of collected data, we investigated a number of fundamental heart rate variability indices and their dependence on age. Therefore, heart beats were extracted and analyzed using the freely available PhysioNet Cardiovascular Signal Toolbox implemented in MATLAB (R2019a, The Mathworks, Natick, MA, USA). As exemplification, standard deviation of heart beat intervals (SDNN), root mean squared difference of successive heart beat intervals (RMSSD), and deceleration capacity (DC) was assessed using the PhysioNet Cardiovascular Signal Toolbox [bib_ref] An Open Source Benchmarked Toolbox for Cardiovascular Waveform and Interval Analysis, Vest [/bib_ref]. For pre-processing of heart beat interval time series, we used an adaptive filter algorithm that is also available online [bib_ref] Nonlinear analysis of complex phenomena in cardiological data, Wessel [/bib_ref]. Outliers in age bins were excluded when exceeding three times the median absolute deviation 18 .
## Measurement protocol.
Measurements were performed in our autonomic laboratory that was temperature controlled at 22 °C. During recordings, the environment was quiet and fully shaded. The illumination level was kept constant via an indirect light source. The recording session started with an interview of the participant. Then, the purpose and design of the study was explained. All study volunteers gave his or her written consent to participate. Weight was measured using a person scale (SECA GmbH & Co. KG., Hamburg, GER) and height using a stadiometer (Harpenden, Holtain Ltd., Crosswell, UK).
After participants lied down comfortably on the examination tilt table, electrodes and pressure cuffs were placed. For the resting state recording, participants were instructed to avoid movement, yawning or coughing.
The instructor waited a few minutes for the participant to calm down and checked the quality of the acquired signals. In case of insufficient signal quality, electrodes and cuffs were re-arranged. Otherwise, the recording was started. The length of the recording was 8 to 35 minutes and was supervised by the instructor. www.nature.com/scientificdata www.nature.com/scientificdata/
Mean ± SD Missing Mean ± SD Missing Mean ± SD Missing Age
## Data records
Data are available at PhysioNet [bib_ref] Components of a new research resource for complex physiologic signals, Goldberger [/bib_ref]. Each recording is stored as a data file with signals of ECG and blood pressure. Blood pressure values were spline-interpolated and resampled to 1000 Hz. Additional information can be found in subject-info.csv that is included in the repository.
## Data anonymization. data was anonymized using the free open-source data anonymization tool arx 21 .
To assure that none of our participants can be identified based on demographic information, we generalized individual age to age groups. The applied a k = 2 anonymity condition and an average re-identification risk of 5% [bib_ref] A Model for Protecting Privacy, Sweeney [/bib_ref]. Files were pseudonymized to numbers 0001-1121 after random ordering.
Recorded data of each participant are stored in a data file (.dat) with raw signals of ECG and blood pressure. Amplitudes are included as a sequence of unsigned 16-bit integers for each signal. An associated header file is an ASCII text file storing meta information on this recording. The first line gives file name, number of signals, sampling frequency and number of samples separated by spaces (e.g., first line in 0023.hae: '0023 2 1000 1226975'). Each of the following lines include information regarding one signal, namely, file name, defined bit resolution, bit gain, baseline in brackets followed by a slash, units, bit resolution followed by a zero, first bit, checksum followed by a zero and signal name (e.g., second line in 0023.hae: '0023.dat 16 10688.4581(−12269)/mV 16 0 −13073 17059 0 ECG'). There is a script to convert wfdb files to mat format (and reverse) in the Waveform Database Software Package for MATLAB (wfdb2mat.m) available at PhysioNet 24 .
The table subjects.tsv gives basic demographic information i.e., age bin, gender and body mass index as well as length of recording in minutes and recording device.
## Technical validation
To explore validity of recorded data, the sample was divided into age bins of ten years (see [fig_ref] 2 Scientific: Data | [/fig_ref]. We estimated standard deviation of heart beat intervals (SDNN) and root mean square of successive differences between heart beat intervals (RMSSD), and deceleration capacity of heart beat intervals. Global heart rate variability as indicated by SDNN seems to decline progressively with advancing age [fig_ref] 2 Scientific: Data | [/fig_ref]. DC and RMSSD suggested that short-term modulation of heart rate decreases until about 50 years of age in a linear matter. At a higher age both parameters remain nearly constant. , and the right abdomen (black). Two ECG channels have been recorded between the electrodes red and yellow (lead I), and between red and green (lead II). Pressure cuffs have been attached around the left arm as well as left middle and index finger. Top right: Two ECG leads were sampled synchronously at 1000 Hz and the continuous blood pressure signal at 100 Hz. Bottom left: In System 2, three electrodes have been attached below the centre of the right clavicle (white), at the left lower thorax (7th intracoastal space at midclavicular line) (red), and at the sternum (black). One ECG channel has been recorded between the electrodes red and white (lead II). Pressure cuffs were attached around the left arm as well as left middle and index finger. Bottom right: The ECG was sampled synchronously at 1000 Hz and the continuous blood pressure signal at 100 Hz. Pictures from www. cnsystems.com and www.biopac.com adapted with permission. www.nature.com/scientificdata www.nature.com/scientificdata/ 23. Dankar, F. K., El Emam, K., Neisa, A. & Roffey, T. Estimating the re-identification risk of clinical data sets. BMC Med. Inform. Decis.
Mak. 12,
[fig] 2 Scientific: Data | (2022) 9:95 | https://doi.org/10.1038/s41597-022-01202-y [/fig]
[fig] Figure 1: Cardiovascular recordings using system 1 -the Task Force Monitor (CNSystems), or system 2 -the CNAP 500 (CNSystems) combined with an MP150 acquisition unit (BIOPAC Systems Inc.). Top left: In System 1, four electrodes have been attached at the right arm (red), the left arm (yellow), the left abdomen (green) [/fig]
[fig] Figure 2: Age dependency of heart rate variability. (a) Standard deviation of heart beat intervals in age bins (SDNN). (b) Root mean square of successive differences between heart beat intervals in age bins (RMSSD). (c) Deceleration capacity of heart beat intervals in age bins (DC). [/fig]
|
Baduanjin Exercise for Stroke Rehabilitation: A Systematic Review with Meta-Analysis of Randomized Controlled Trials
Objective: The purpose of this review was to objectively evaluate the effects of Baduanjin exercise on rehabilitative outcomes in stroke patients. Methods: Both Chinese and English electronic databases were searched for potentially relevant trials. Two review authors independently screened eligible trials against the inclusion criteria, extracted data, and assessed the methodological quality by using the revised PEDro scale. Meta-analysis was only performed for balance function. Results: In total, there were eight randomized controlled trials selected in this systematic review. The aggregated result of four trials has shown a significant benefit in favor of Baduanjin on balance function (Hedges' g = 2.39, 95% CI 2.14 to 2.65, p < 0.001, I 2 = 61.54). Additionally, Baduanjin exercise effectively improved sensorimotor function of lower extremities and ability of daily activities as well as reduced depressive level, leading to improved quality of life. Conclusion: Baduanjin exercise as an adjunctive and safe method may be conducive to help stroke patients achieve the best possible short-term outcome and should be integrated with mainstream rehabilitation programs. More rigorous randomized controlled trials with long-term intervention periods among a large sample size of stroke patients are needed to draw a firm conclusion regarding the rehabilitative effects for this population.
# Introduction
Stroke (also called cerebrovascular disease) is a devastating medical condition in which cerebral vascular dysfunction suddenly occurred, leading to cell deaths. This neurological disorder is caused by cerebral blood flow and cerebral vascular occlusion (ischemic stroke) or cerebrovascular rupture (hemorrhagic stroke). Sensorimotor impairments (e.g., losses of body sensation and proprioception, and altered patterns of coordination and balance) are commonly reported among survivors of stroke, which is directly associated with inability to initiate voluntary movement or hemiplegia. In addition, depressive symptom is a common and serious complication among post-stroke survivors; roughly 30% of post-stroke survivors develop depression, which may be attributed to feeling hopeless about their future lives. Such stroke-induced symptoms have greatly affected activities of daily living and reduced quality of life.
Globally, 15 million individuals suffer from a stroke each year, with 5.5 million deaths and 5 million with a loss of their capabilities for independent living. An epidemiologic study published in 2007 reported that the number of stroke cases rapidly increased in China and had reached more than 7 million; roughly 70-80% of individuals survive who lost functional abilities and require treatment to improve the stroke-related symptoms. In the United States, more than 700,000 individuals suffer a stroke annually and roughly two-thirds of these post-stroke survivors require rehabilitation. Available evidence indicates that conventional rehabilitation techniques (cycle-ergometer, rising from a chair) and device-assisted therapy (virtual reality) are effective for post-stroke survivors. However, it is noted that these rehabilitation methods are time-consuming and costly, which may preclude post-stroke survivors with low social-economic status. In addition, these rehabilitations have simply emphasized physical training for improving sensorimotor function; the mental illness (depression, anxiety, and mood disturbance) in post-stroke survivors could not be alleviated. Therefore, searching for more cost-effective and readily accessible approaches is critically demanded for improving both physical and psychological symptoms among post-stroke survivors.
Baduanjin, as one of the traditional Chinese health-promoting exercises (e.g., Tai Chi, Baduanjin, Wuqinxi), was originally created to help soldiers recover from bodily injuries in ancient China and prepare for the next battle. Baduanjin contains eight simple postures and movements, which makes it readily accessible to a variety of people. Additionally, when compared to other conventional physical exercises, Baduanjin training does not simply improve musculoskeletal and neuromuscular function, but its unique features (mind-body relaxation, mental focus, and breathing control) may also make post-stroke survivors feel more pleasant and enhance their adherence to Baduanjin exercise for psychological well-being. Baduanjin has become more popular since the in 2002 Chinese Health-Qigong Association was established in 2002 to introduce this mindful exercise to people around the world, which has naturally attracted attention from the research community. Although the number of trials were conducted to investigate the effects of Baduanjin exercise on rehabilitative outcomes in post-stroke survivors, to date no systematic review has been done to synthesize these research findings. Thus, we conducted a systematic review to objectively evaluate the existing literature relating to the rehabilitative effects of Baduanjin for post-stroke survivors. The updated information of this review would allow future researchers and clinicians to design and develop effective mind-body exercise protocols for accelerating the psychosomatic rehabilitation of post-stroke survivors.
# Methods
## Data sources
We used the following databases for literature search without restricting date of publication: PubMed, Cochrane Library, WHO International Clinical Trials Registry Platform, Science Direct, Allied and Complementary Medicine Database, China National Knowledge Infrastructure, Wanfang, and Chinese Clinical Trial Registry. To ensure inclusion of all relevant trials, relevant search terms were combined with Boolean conjunction (OR/AND) and used according to three search levels: (i) Baduanjin or Eight Brocade Section; (ii) stroke, cerebrovascular accident, or cerebrovascular disease; (iii) rehabilitation, therapy, balance, functional status, sensorimotor function, or mental disorder. Cross-reference search was also used to manually identify relevant trials. The detailed information of this systematic review and meta-analysis is reported following the Preferred Repointing items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines.
## Inclusion criteria and study selection
Randomized controlled trials were published in a peer-reviewed English or Chinese journal and investigated the rehabilitative impacts of Baduanjin for stroke survivors. Trials in which there was at least one pairwise comparison between an experimental group that received Baduanjin exercise as the main intervention and a passive (unaltered lifestyle or waitlist) or active control (walking, drug therapy, or balance training) group. Rehabilitative outcomes were considered in this systematic review, including sensorimotor function, balance, abilities of daily living, and psychological parameters (e.g., depression or anxiety), and quality of life. Documents excluded involved observational studies, case studies, case reports, unpublished thesis and dissertation, and conference proceeding.
## Methodological quality assessment of eligible studies
The revised Physiotherapy Evidence Database (PEDro) scale was used to assess the study quality of trials selected. The revised PEDro contains 10 items: random assignment, concealed allocation, similar baseline, blinding of assessors, more than 85% retention, missing data management, between-group comparison, point measure and measure of variability, isolated Baduanjin intervention, and prior sample size calculation. Blinding of participants and instructor(s) were not considered because they are impractical in exercise intervention studies. Each study can obtain a sum score ranging from zero to ten, with higher scores indicating better study quality.
## Data extraction and analysis
All identified trials were reviewed by two independent authors (Liye Zou and Chaoyi Wang). The detailed information from eligible trials was extracted, including reference (name of leading author, year of publication, and type of experimental design), location (language), participant characteristics (sample size and dropout rate, mean age/age range of study participants, course of disease, and type of stroke), intervention program (training duration and intensity), outcome measured, study findings, adverse event, and follow-up assessment. Because the heterogeneity of outcome measures in a small number of randomized controlled studies, meta-analysis (Comprehensive Meta-Analysis Version 2.0 software; Biostat, NJ, USA) was only performed on balance function. While the fixed-effect model was used, effect size (Hedge's g) was used to evaluate the magnitude of the effect of Baduanjin on balance function in stroke patients. The value of Hedges' g can be interpreted based on the following rule of thumb: small effect = 0.2, medium effect = 0.5, and large effect = 0.8.
# Results
## Study selection
Literature search resulted in 175 potentially relevant articles. After removing 143 duplicates, 32 records remained. When reading the titles and abstract of the remaining studies, 15 irrelevant records were identified and removed. This is followed by full-text article evaluation and nine full-text articles were excluded (non-Baduanjin as the main intervention = 1; reviews = 3; No interesting outcome = 3; study protocol = 2). Finally, a total of 8 randomized controlled trials was left in this systematic review.
## Study characteristics
Eight randomized controlled trials were conducted between 2010 and 2017 in China. All trials in which there were 822 older adults with stroke (sample size in individual trials ranging from 60 to 224). Course of disease among these post-stroke survivors was reported ranging from one to six months. In total, there were 269 participants with ischemic stroke and 105 with hemorrhage stroke in five trials (other two trials did not report the type of stroke). Baduanjin intervention program (training duration ranged from 40 days to 12 weeks) varied from 30 to 80 min per day, two to seven times weekly. Group-based Baduanjin training was carried out in all clinical trials by certified instructors. Baduanjin as the main intervention was combined with educational program, general rehabilitation, Bobath technique and drug therapy, or balance training and general rehabilitation. Post-stroke patients in the active control groups had received educational program, general rehabilitation, Bobath technique and drug therapy, or balance training and general rehabilitation.
## Study characteristics
Eight randomized controlled trials were conducted between 2010 and 2017 in China. All trials in which there were 822 older adults with stroke (sample size in individual trials ranging from 60 to 224). Course of disease among these post-stroke survivors was reported ranging from one to six months. In total, there were 269 participants with ischemic stroke and 105 with hemorrhage stroke in five trials (other two trials did not report the type of stroke). Baduanjin intervention program (training duration ranged from 40 days to 12 weeks) varied from 30 to 80 min per day, two to seven times weekly. Group-based Baduanjin training was carried out in all clinical trials by certified instructors. Baduanjin as the main intervention was combined with educational program, general rehabilitation, Bobath technique and drug therapy, or balance training and general rehabilitation. Post-stroke patients in the active control groups had received educational program, general rehabilitation, Bobath technique and drug therapy, or balance training and general rehabilitation.
## Study quality
The methodological quality of all trails selected ranged from five to seven, with a higher score indicating lower risk of bias. Points in all trials were deducted due to the lack of concealed allocation and isolated Baduanjin intervention and prior sample size estimation. This is followed by deducted points on blinding of assessors in five trialsand missing data management in three trials. Note: Item 1 = randomization; Item 2 = concealed allocation; Item 3 = similar baseline; Item 4= blinding of assessors; Item 5 = more than 85% retention; Item 6 = missing data management (intention-to-treat analysis); Item 7 = between-group comparison; Item 8 = point measure and measures of variability; Item 9 = isolated Baduanjin intervention; Item 10 = prior sample size estimation;1 = explicitly described and present in details; 0 = absent, inadequately described, or unclear.
## Effects of baduanjin on stroke-related outcomes
For meta-analysis, four trials investigated effects of Baduanjin on balance function, as measured by the Berg Balance Scale. A higher positive value indicates better balance function. The aggregated result has shown a significant benefit in favor of Baduanjin on balance function (a large effect size, but moderate heterogeneity: Hedges' g = 2.39, 95% CI 2.14 to 2.65, p < 0.001, I 2 = 61.54).
## Study quality
The methodological quality of all trails selected ranged from five to seven, with a higher score indicating lower risk of bias. Points in all trials were deducted due to the lack of concealed allocation and isolated Baduanjin intervention and prior sample size estimation. This is followed by deducted points on blinding of assessors in five trialsand missing data management in three trials. Note: Item 1 = randomization; Item 2 = concealed allocation; Item 3 = similar baseline; Item 4= blinding of assessors; Item 5 = more than 85% retention; Item 6 = missing data management (intention-to-treat analysis); Item 7 = between-group comparison; Item 8 = point measure and measures of variability; Item 9 = isolated Baduanjin intervention; Item 10 = prior sample size estimation;1 = explicitly described and present in details; 0 = absent, inadequately described, or unclear.
## Effects of baduanjin on stroke-related outcomes
For meta-analysis, four trials investigated effects of Baduanjin on balance function, as measured by the Berg Balance Scale. A higher positive value indicates better balance function. The aggregated result has shown a significant benefit in favor of Baduanjin on balance function (a large effect size, but moderate heterogeneity: Hedges' g = 2.39, 95% CI 2.14 to 2.65, p < 0.001, I 2 = 61.54). Sensorimotor function and neurological deficit: two trials assessed sensorimotor function on lower extremity with the Fugl-Meyer Assessment-Lower Extremity. Study findings from the two trials indicated significant improvement on lower extremity-related sensorimotor function. Specifically, Tian et al.considered Baduanjin + general rehabilitation as an experimental group, whereas general rehabilitation alone was thought of as a control group. After a 12-week intervention period, Baduanjin group (12.45 ± 7.24 vs. 23.31 ± 7.63) showed significantly greater improvement on sensorimotor function of lower extremities than the control group (13.37 ± 6.55 vs. 17.13 ± 5.72). Another trial in which researchers compared an experimental group (Baduanjin + balance training + general rehabilitation) with a control group (balance training + general rehabilitation) suggested that Baduanjin (12.3 ± 3.56 vs. 27.18 ± 5.11) also had significantly greater improvement on sensorimotor function of lower extremity than the control group (12.44 ± 3.2 vs. 21.17 ± 4.36). Additionally, Guo et al.investigated the effects of Baduanjin on neurological function, as measured by National Institute of Health Stroke Scale (a higher score indicating worse symptom), suggesting that Baduanjin group (15.97 ± 2.84 vs. 5.25 ± 2.54) showed significantly greater improvement than the control group (16.17 ± 2.76 vs. 7.66 ± 2.49).
Depression, activities of daily living, and quality of life: two trials assessed quality of life with two different self-reported questionnaires. Caireported that significant improvements on physical, psychological, and environmental domains of World Health Organization Quality of Life were only observed in Baduanjin group (p < 0.05) from baseline to Week-12, but not in the control group. Another trial by Chen et al.in which both groups received Baduanjin practice combined with different components (general rehabilitation vs. general rehabilitation + music) suggested that significant improvement on the sum score of quality of life, as measured by the SF-36. As compared to the general rehabilitation group, Baduanjin with music was more beneficial for improving quality of life and eight individual domains (vitality, physical functioning, bodily pain, general health perceptions, physical role function, emotional role functioning, and mental health). Meanwhile, Chen et al.also assessed depressive symptom (with the Hamilton Depression Scale) among post-stroke survivors, suggesting that Baduanjin effectively reduced depression in both groups, with greater reduction of depression in a combined group (Baduanjin + music + general rehabilitation). The ability of daily activities was assessed using the Barthel Scale in a trial in which an experimental group receiving a combination of Baduanjin and an education program was compared with those people in a control group who received the same educational program alone. Cai et al.reported significant improvement in the Barthel Scale in the Baduanjin group (p < 0.001), but not in the control group.
# Discussion
The purpose of this systematic review was to critically evaluate the effects of Baduanjin exercise on rehabilitative outcomes among post-stroke survivors. The study findings from this present review suggest that Baduanjin exercise may be a promising approach for improving functional capabilities of lower extremities (static and dynamic balance and sensorimotor function) and alleviating depressive symptoms and reducing neurological deficit scores. Baduanjin exercise may be also conducive to improving quality of life and the ability of daily activities among survivors of stroke.
Baduanjin routine involves symmetrical postures and coordinated movements, which requires practitioners to sustainably maintain the balance with weight-shifting movement or moving their arms, legs, and torso to change the center of gravity. For example, in performing Movement 5 ("sway the head and shake the tail"), the practitioner needs to squat in a low horse stance while placing the hands on the thighs with the elbows facing out, followed by drawing a circle with his or her upper body (shift his or her weight of the upper body on each side) without losing his or her balance. Thus, it is reasonable that Baduanjin exercise has a significant effect on improving balance function and sensorimotor function of lower extremities among post-stroke survivors. Such these study findings of the present review are supported by previous studies investigating the rehabilitative effects of Baduanjin for functional abilities of lower extremities in vulnerable populations, particularly in patients with Parkinson's disease who are typically facing balance function impairment. Baduanjin does not only focus on strengthening physical function, but also has emphasized mental practice to coordinate with breathing technique and musculoskeletal relaxation.found that mindfulness-based Baduanjin exercise effectively elevated adiponectin level, which potentially contributed to reduced depression in patients with chronic disease. It may be a reason the depressive symptom in post-stroke survivors was alleviated. Since these aforementioned outcomes were improved, the ability of daily activities and quality of life of post-stroke survivors may be improved accordingly.
Despite the promising results were found in the present review, several limitations must be acknowledged. First, all trials selected had a higher risk of bias in this systematic review, which is due to lack of concealed allocation, blinding of assessors, missing data management, and prior power analysis. In particular, one point was deducted in all trials because Baduanjin exercise was combined with other mainstream rehabilitation programs, meaning that it may be difficult to determine whether the positive outcomes were due to Baduanjin alone or synergetic effects. It must be admitted that combined intervention is legitimate because the ultimate goal of Baduanjin exercise as an adjunctive method is to help survivors become as independent as possible and to attain the best possible quality of life. Second, two-thirds of the trials selected involved short duration of intervention without follow-up assessment, so the study findings from the existing literature are limited to the immediate effects of Baduanjin. Third, because of the limited number of studies investigating the effects of Baduanjin on sensorimotor function and neurological function, the ability of daily activities, and quality of life, it remains unclear whether these outcomes were improved by chance. Fourth, all trials were conducted in China and the stroke patients were predominantly Chinese. It is unclear whether the improved balance is generalizable to non-Chinese populations. Fifth, although meta-analysis with RCTs is advantageous, given the fact the study design allows for the determination of the effect of an intervention, it actually decreases the pool of studies from which the authors could gather data; in particular, only 8 articles were included in the current study. For example, other experimental studies (e.g., controlled trials with no randomization and pre-test and post-test studies) may be able to partially explain the relationship between Baduanjin exercise and rehabilitative outcomes.
To better evaluate the effects of Baduanjin exercise on rehabilitative outcomes in patients with stroke, more high-quality studies with random design should be conducted, particularly considering concealed allocation, blinded assessors, intent-to-treat analysis, and sample size calculation. Follow-up period should be considered after an intervention to determine the long-term effects of Baduanjin exercise for stroke rehabilitation. Isolating other treatments or co-intervention will allow for better explanation of the stand-alone benefits that Baduanjin exercise may have, as compared to either active or passive control. Finally, non-Chinese people with stroke should be investigated to determine if Baduanjin exercise is suitable and beneficial for other races with stroke.
# Conclusions
Baduanjin exercise as a safe and adjunctive rehabilitation method might have beneficial effects for balance function, sensorimotor function of lower extremities, depression, the ability of daily activities, and quality of life in survivors of stroke. However, the small number of trials with methodological flaws in their design makes a definitive conclusion hard to be drawn. More well-designed randomized controlled trials with larger sample size and longer intervention periods are needed to establish the effects of Baduanjin on rehabilitative outcomes among stroke patients. In addition, strong scientific evidence could not be obtained because we could not eliminate bias: (1) It has not received international evaluation in the articles because these papers are all Chinese characters; (2) the authors are often overlapped eight extracted papers, and independent papers are regarded as three papers as research; (3) the four selected studiesinvestigated the effects of Baduanjin exercise on balance function, as measured by BBS, two of the studies were conducted by a same research team. Based on the aforementioned limitations, at the present stage, there is publication bias. The bias is that published papers are limited to Chinese language only. There is also publication bias such as limitation of author, and few pieces of independent research. For reasons such as this, the effect of Baduanjin exercise is expected to be limited. At the present stage, because of the meta-analysis, although the effect was recognized, it is very weak as a scientific basis. Thus, the research on Baduanjin exercise will continue to accumulate. |
Prospective mapping of viral mutations that escape antibodies used to treat COVID-19
Antibodies are a potential therapy for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but the risk of the virus evolving to escape them remains unclear. Here we map how all mutations to the receptor binding domain (RBD) of SARS-CoV-2 affect binding by the antibodies in the REGN-COV2 cocktail and the antibody LY-CoV016. These complete maps uncover a single amino acid mutation that fully escapes the REGN-COV2 cocktail, which consists of two antibodies, REGN10933 and REGN10987, targeting distinct structural epitopes. The maps also identify viral mutations that are selected in a persistently infected patient treated with REGN-COV2 and during in vitro viral escape selections. Finally, the maps reveal that mutations escaping the individual antibodies are already present in circulating SARS-CoV-2 strains. These complete escape maps enable interpretation of the consequences of mutations observed during viral surveillance.
A ntibodies are being developed as therapeutics to combat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (1). Antibodies against some other viruses can be rendered ineffective by viral mutations that are selected during treatment of infected patientsor that spread globally to confer resistance on entire viral clades. Therefore, determining which SARS-CoV-2 mutations escape key antibodies is essential for assessing how mutations observed during viral surveillance may affect the efficacy of antibody treatments.
Most leading anti-SARS-CoV-2 antibodies target the viral receptor binding domain (RBD), which mediates binding to the angiotensinconverting enzyme 2 (ACE2) receptor. We recently developed a deep mutational scanning method to map how all mutations to the RBD affect its function and recognition by antiviral antibodies. This method involves creating libraries of RBD mutants, expressing them on the surface of yeast, and using fluorescenceactivated cell sorting and deep sequencing to quantify how each mutation affects RBD folding, ACE2 affinity (measured across a titration series), and antibody binding (fig. S1A). In this study, we used the duplicate mutant libraries described in, which consist of barcoded RBD variants that cover 3804 of the 3819 possible amino acid mutations. Our libraries were made in the genetic background of the RBD from the early isolate Wuhan-Hu-1, which still represents the most common RBD sequence, although several mutants are currently increasing in frequency. We mapped how the 2034 mutations that do not strongly disrupt RBD folding and ACE binding (7) affected binding by recombinant forms of the two antibodies in Regeneron's REGN-COV2 cocktail (REGN10933 and REGN10987)and Eli Lilly's LY-CoV016 antibody (also known as CB6 or JS016) (13) (fig. S1B). REGN-COV2 was recently granted emergency use authorization for treatment of COVID-19, whereas LY-CoV016 is currently in phase 3 clinical trials.
We completely mapped RBD mutations that escape binding by the three individual antibodies as well as the REGN-COV2 cocktailand B, and zoomable maps at https://jbloomlab.github.io/SARS-CoV-2-RBD_MAP_clinical_Abs/). REGN10933 and REGN10987 are escaped by largely nonoverlapping sets of mutations in the RBD's receptor binding motif, consistent with structural work showing that these antibodies target distinct epitopes in this motif. But unexpectedly, one mutation [Glu 406 → Trp (E406W)] strongly escapes the cocktail of both antibodies. The escape map for LY-CoV016 also reveals escape mutations at a number of different sites in the RBD. Although some escape mutations impair the RBD's ability to bind ACE2 or be expressed in properly folded form, many come at little or no cost to these functional properties, according to prior deep mutational scanning measurements using yeast-displayed RBD (7) (color gradient in, A and B, indicates loss of ACE2 affinity and in indicates decrease in RBD expression).
To validate the antigenic effects of key mutations, we performed neutralization assays using spike-pseudotyped lentiviral particles and found concordance between the antibody binding escape maps and neutralization assays. As expected from the maps for the REGN-COV2 antibodies, a mutation at site 486 escaped neutralization only by REGN10933, whereas mutations at sites 439 and 444 escaped neutralization only by REGN10987-and so none of these mutations escaped the cocktail. But E406W escaped both individual REGN-COV2 antibodies and thus also strongly escaped the cocktail. Structural analyses and viral-escape selections led Regeneron to posit that no single amino acid mutation could escape both antibodies in the cocktail, but our complete maps identify E406W as a cocktail escape mutation. E406W affects the REGN-COV2 antibodies in a relatively specific way and does not grossly perturb the function of the RBD, given that it only mildly reduces neutralization by LY-CoV016and the titers of spike-pseudotyped lentiviral particles .
To explore whether our escape maps are consistent with how the virus evolves under antibody selection, we first examined data from Regeneron's viral escape-selection experiments in which spike-expressing vesicular stomatitis virus (VSV) was grown in cell culture in the presence of either REGN10933, REGN10987, or the REGN-COV2 cocktail. That work identified five escape mutations from REGN10933, two from REGN10987, and none from the cocktail . All seven cell culture-selected mutations were prominent in our escape maps while also being accessible by just a single-nucleotide change to the wild-type codon in the Wuhan-Hu-1 RBD sequence , demonstrating concordance between the escape maps and viral evolution under antibody pressure in cell culture. Notably, E406W is not accessible by a singlenucleotide change, which may explain why it was not identified by the Regeneron cocktail selections despite being relatively well tolerated for RBD folding and ACE2 affinity.
To determine whether the escape maps could inform analysis of viral evolution in infected humans, we examined deep sequencing data from a persistently infected immunocompromised patient who was treated with REGN-COV2 at day 145 after diagnosis with COVID-19. The late administration of the treatment allowed ample time for the patient's viral population to accumulate genetic diversity, some of which could have been driven by immune pressure, because the patient mounted a weak autologous neutralizing antibody response before treatment. Administration of REGN-COV2 was followed by rapid changes in the frequencies of five amino acid mutations in the RBD. Our escape maps showed that three of these mutations escaped REGN10933 and that one escaped REGN10987 . Notably, the mutations Sites of strong escape (purple underlines) are shown in logo plots at right. The height of each letter is proportional to how strongly that amino acid mutation mediates escape, with a per-mutation "escape fraction" of 1 corresponding to complete escape. The y-axis scale is different for each row, so, for instance, E406W escapes all REGN antibodies but is most visible for the cocktail as it is swamped out by other sites of escape for the individual antibodies. See https://jbloomlab.github.io/SARS-CoV-2-RBD_MAP_clinical_Abs/ for zoomable versions. Letters are colored according to how mutations affect the RBD's affinity for ACE2 as measured via yeast display (7) For each antibody or antibody combination, the escape score for each mutation is plotted versus its frequency among the 317,866 high-quality human-derived SARS-CoV-2 sequences on GISAID (26) as of 11 January 2021. Escape mutations with notable GISAID frequencies are labeled. The REGN-COV2 cocktail escape mutation E406W requires multiple nucleotide changes from the Wuhan-Hu-1 RBD sequence and is not observed among sequences in GISAID. Other mutations to residue E406 (E406Q and E406D) are observed with low frequency counts, but neither of these mutant amino acids is a single-nucleotide mutation away from W either.
did not all sweep to fixation after antibody treatment; instead, there were competing rises and falls . This pattern has been observed in the adaptive within-host evolution of other virusesand can arise from genetic hitchhiking and competition among viral lineages. Both these forces appear to be at play in the persistently infected patient: E484A (not an escape mutation in our maps) hitchhikes with F486I (which escapes REGN10933) after treatment, and the viral lineage carrying N440D and Q493K (which escape REGN10987 and REGN10933, respectively) competes first with the REGN10933 escapemutant Y489H and then with the lineage carrying E484A and F486I and the Q493K lineage. Three of the four escape mutations in the REGN-COV2-treated patient were not identified in Regeneron's viral cell culture selections , illustrating an advantage of complete maps. Viral selections are incomplete in the sense that they only identify whatever mutations are stochastically selected in that particular cell culture experiment. In contrast, complete maps annotate all mutations, which could include mutations that arise for reasons unrelated to treatment but incidentally affect antibody binding.
Of course, viral evolution is shaped by functional constraints as well as pressure to evade antibodies. The mutations selected in cell culture and the patient consistently met the following criteria: They escaped antibody binding, were accessible via a singlenucleotide change, and imposed little or no cost on ACE2 affinity [as measured by prior deep mutational scanning using yeast-displayed RBD (7)] . Therefore, com-plete maps of how mutations affect key biochemical phenotypes of the RBD (e.g., ACE and antibody binding) can be used to assess likely paths of viral evolution. One caveat is that over longer evolutionary time frames, the space of tolerated mutations could shift as a result of epistatic interactions, as has been observed in viral immune and drug escape.
The complete maps enable us to assess what escape mutations are already present among circulating SARS-CoV-2. We examined all human-derived SARS-CoV-2 sequences available as of 11 January 2021 and found a substantial number of RBD mutations that escaped one or more of the antibodies . However, the only escape mutations present in >0.1% of sequences were the REGN10933 escapemutant Y453F [0.3% of sequences; see also], the REGN10987 escape-mutant N439K . Y453F is associated with independent outbreaks linked to mink farms in the Netherlands and Denmark; notably, the mink sequences themselves sometimes also contain other escape mutations, such as F486L. N439K is prevalent in Europe, where it has constituted a large percentage of sequences from regions including Scotland and Ireland. K417N is present in the B.1.351 lineage first identified in South Africa. Another mutation of current interest is N501Y, which is present in B.1.351 and also the B.1.1.7 lineage originally identified in the United Kingdom (9). Our maps indicate that N501Y has no effect on either of the REGN-COV2 antibodies and has only a modest effect on LY-CoV016 .
To determine whether the escape maps could be rationalized from the structural interfaces of the antibodies and RBD, we projected the maps onto crystal or cryo-electron microscopy structures; interactive versions at https:// jbloomlab.github.io/SARS-CoV-2-RBD_MAP_ clinical_Abs/). As might be expected, escape mutations generally occur in the antibody-RBD interface. However, structures alone are insufficient to predict which mutations mediate escape. For example, LY-CoV016 uses both its heavy and light chains to bind a wide epitope overlapping the ACE2-binding surface, but escape is dominated by mutations at RBD residues that contact the heavy-chain complementarity-determining regions, E to G). In contrast, escape from REGN10933 and REGN10987 mostly occurs at RBD residues that pack at the antibody heavyand light-chain interface, A to D). The E406W mutation that escapes the REGN-COV2 cocktail occurs at a residue not in contact with either antibody. Although E406 is in closer structural proximity to LY-CoV016, the E406W mutation has a much smaller impact on this antibody, suggesting a long-range structural mechanism specific to the REGN-COV2 antibodies . Taken together, mutations at RBD residues that contact antibody do not always mediate escape, and several prominent escape mutations occur at residues not in contact with antibody.
In this study, we have completely mapped mutations that escape three leading anti-SARS-CoV-2 antibodies. These maps demonstrate that prior characterization of escape mutations was incomplete, having identified neither a single amino acid mutation that escapes both antibodies in the REGN-COV2 cocktail nor most mutations that arose in a persistently infected patient treated with the cocktail. Of course, our maps still do not answer the most pressing question: Will SARS-CoV-2 evolve widespread resistance to these antibodies? But certainly, it is concerning that so many escape mutations impose little cost on RBD folding or receptor affinity and that some are already present at low levels among circulating viruses. Ultimately, it will be necessary to wait and see what mutations spread as SARS-CoV-2 circulates in the human population. Our work will help with the "seeing," by enabling immediate interpretation of the effects of the mutations cataloged by viral genomic surveillance. |
Hematopoietic progenitors polarize in contact with bone marrow stromal cells in response to SDF1
[formula] --------------------------------------------------------------------------- [/formula]
## Editors
You will see that all the reviewers found the observations that HSPCs interact with various niche cells through a contact interface that involves polarization exciting. They made several suggestions to better understand the molecular basis of the interactions and their relevance. From their comments, we suggest you focus a revision on the following points: 1) We agree with Reviewer #1 that testing primary T cells, as opposed to Jurkat, would strengthen the work and be consistent with the other analyses presented. These studies would bolster your conclusion that "hematopoietic polarization is not a generic outcome but specific to defined interactions between hematopoietic progenitors and stromal cells". Similarly, we encourage you to add primary myeloid cells, such as a monocyte or granulocyte, too. Better testing the cell type specificity of the interactions would deepen the understanding of their role and provide a stronger test to your overall model.
We followed your recommendation and tested the polarity of human primary T cells (CD4+, activated and non-activated) and monocytes (CD14+) on osteoblasts (new . None of them were polarized, thus confirming that this property was specific to HSPC.
2) On a related note, Reviewer #1 suggests looking at the distribution of SDF1 and CXCR4; this should be straightforward to do and would nail down just what cell types and lineages will be under this mechanism of development.
We performed these measurements with immuno-fluorescence and western-blots. With the western blots, we found that SDF1 is more expressed in stromal cells of the bonemarrow (osteoblast and endothelial cells), which can induce the polarization of HSPC, than on fibroblast, which can not induce their polarization (new . With immunofluorescence we found that SDF1 and CXCR4 are concentrated in but not limited to the point of interaction between HSPCs and osteoblasts. They were also present in vesicles located in close vicinity with the contact site (new .
3) Please address Reviewer #3's points in full. In particular, the reviewer cites earlier work that questions whether the comparison to an IS is warranted. Rethinking the stalks in this context vs the immune synapse context is important, as Reviewer #1 also suggests in point #2.
We apologize for the lack of clarity and the misunderstanding due to the fact that we did not named the structure properly. We now refer to the polarized structure as the "magnupodium", a term that was proposed in earlier studies on HSPC (that were already cited in our initial manuscript). It refers to a structure that is closer to the one we investigated than the stalk described in T cells. We think the comparison of the magnupodium to the uropod and the immune synapse is relevant and useful. First because these are the two polarized structures of reference that have been described in the blood lineage. Both could be related to the magnupodium since they are all associated with the centrosome. But the uropod stands in the back of a moving cell, whereas the immune synapse is at the front of an anchored cell, so it is important to know to which one the magnupodium is closer. Second because the magnupodium has been lately considered to be closer to the uropod, as it was described in moving HSPC, while we rather think it is closer to the immune synapse. We paid attention not to confuse the magnupodium with the immune synapse and clarified this in the discussion.
However we kept open the possibility that it may be considered as an "hematopoietic synapse" in the future. 4) Reviewer #2 raises important and interesting general questions about the basis of the interaction and its physiological role. JCB Reports must present definitive, highly novel observations of outstanding interest to a wide readership that have the potential to open up new avenues of research. Because of the emphasis on novelty and broad interest, Reports do not require the same mechanistic depth as full JCB Articles. Here, your study identifies a new relevant receptor-ligand interaction to trigger contact that may be an important HSPC activity. We feel that the work fulfills the editorial requirements of the format without substantial further mechanistic or functional studies. However, determining whether polarization impacts or is needed for differentiation would certainly be very interesting. If you have such data, we encourage you to add them. If these studies are not already underway, we would not require the data for publication.
We appreciate your understanding. Adrian Candelas started its PhD in 2019 to investigate the potential effect of HSPC polarization on their differentiation. He first duplicated all the key experiments we described in this manuscript and performed all the experiments suggested by the reviewers (primary cells, SDF1 quantification and immunostainings, SDF1 agonist in cell-cell and cell-substrate interactions). He is thus signing the manuscript as a co-first author. He will now focus on its initial project. 5) Lastly, Ira and I have one strong suggestion to increase the impact and relevance of the study: the drug AMD3100/plexafor is a well-known CXCR4 antagonist (CXCR7 agonist) that is used clinically to mobilize HSCs, but its mechanism of action is unclear. If you can obtain the drug and add it to your system to test if it blocks polarization or causes depolarization, it will yield significant insight that will increase the breadth and appeal of your work. It will also be an independent opportunity to confirm your hypothesis that the SDF1/CXCR4 interaction is predominant in generating HSC polarity. Since you do not investigate the functional consequences of polarity induction (e.g., with respect to triggering lineage differentiation), we believe this is an important addition.
We followed your recommendation and used AMD3100 to block CXCR4 interaction with SDF1 in both HSPC-osteoblast and HSPC-ligand assay. In both cases the polarization of HSPC was impaired (new , 5E).
We'd be happy to discuss the revision further if you anticipate any issues addressing the reviewers' comments or have any questions.
## Reviewer #1
The bone marrow, consisting of multiple types of stromal cells and soluble factors (diffusive signals), forms a niche to instruct the fate of HSPCs, which polarize both morphologically and biochemically during differentiation, but the exact cell types and signaling pathways causing HSPCs polarization is unknown. It is also difficult to dissect the contribution of cell migration and cell-cell interaction (anchorage) in a native or conventional assay system.
In the current manuscript, Bessy et al. modeled the bone-marrow niche using an elegant microwell cell-culture system which promote long-term cell-cell interaction but prevents cell migration. This system allowed them to visualize in 3D, long-term interactions between human HSPCs and niche cells at a single cell level. They found that when an HSPC forms a small contact with an osteoblast, both centrosome and actin networks polarize to the contact zone while the nucleus polarizes to the distal end, resembling what was reported for an immunological synapse but not a uropod. The authors then tested the specificity of the HSPC polarization, by coculturing HSPCs with three types of stromal cells: umbilical vein endothelial cells, bone-derived osteoblast and skin fibroblast.
The authors then provided evidence that polarization is cell-type specific. After assessing the cell morphology and centrosome positioning, the authors found that the HSPCs polarize upon contact with umbilical vein endothelial cells or with osteoblasts but not with fibroblasts. They further showed that HSPC polarization was correlated with stromal cell functions: AFT024, a murine stromal cell line known to support HSPC regeneration, induced HSPC polarization, whereas BFC012, a murine stromal cell line that does not support HSPC regeneration, failed to induce HSPC polarization. On the other hand, stromal cells induced polarization of HSPCs but not mature Jurkat T cell line.
Finally, the authors examined which molecular pathway(s) are responsible to HSPC polarization during HSPC-stromal cell contact. Using ligand-coated microwell, they provided evidence that VCAM-VLA4, ICAM-LFA1, or SDF1-CXCR4 pathway is sufficient for HSPCs to polarize morphologically, but only SDF1-CXCR4 pathway is sufficient for centrosome and microtubule polarization.
Overall this is a very nice paper. The study was well designed and executed. The findings are novel and interesting and presented in a logical manner. The methods are unique and powerful. The paper may be improved by providing some further molecular insights.
We thank the reviewer for these positive comments and for the great suggestions of additional experiments which did reinforce our conclusions.
Major:
1. One intriguing aspect of this paper is the cell-type specificity. The authors also suggested that certain cell adhesion pathways, specifically the SDF1-CXCR4 axis, are central for HSPCs polarization. Are these pathways absent or downregulated in the fibroblast-HSPC, and stromal-lymphocyte context? Can the authors analyze the expression of SDF1, VCAM and ICAM in the skin fibroblast, and the expression of CXCR4 in HSPCs and mature lymphocytes? These experiments should enhance the molecular insights of the paper.
We followed reviewer's advice and compared the expression SDF1 in bone-marrow stromal cells and in skin fibroblast. We found that it was much less expressed in skin fibroblast (new , which is consistent with their lower ability to induce the polarization of HSPCs.
2. Formation of immune synapse involving a T cell is thought to require the stimulation of the antigen receptor, so I was left wondered why the HSPC polarization was induced in the absence of antigen. Or is the term "immune synapse" an overstatement as indicated by reviewer #3? This is correct. The polarized structure of HSPC we described is not an immune synapse. We apologize for the lack of clarity. We argued that, based on the localization of specific proteins, it resembles more to an immune synapse than a uropod. Indeed, the polarized structures of HSPC have often been compared to uropod-like structures since they were observed in migrating cells. Here we provide evidences that the polarized structures that HSPCs form in contact with osteoblast or endothelial cells do not contain some of the characteristic markers of uropod, and instead share more features with immune synapse. Our new immunostainings of SDF1 and CXCR4 revealed some vesicles in the vicinity of the contact site, further supporting the possibility that the two cell types are actually exchanging signals and materials. However we agree that the structure we described is not an immune synapse and made this point clear in the revised main text and discussion.
## The authors showed that stromal cells did not induce polarization of jurkat cells. given
Jurkat is a leukemia cell line that lack critical signaling components, such as PTEN, it will be worthwhile to determine whether the finding holds for primary T cells.
We followed reviewer's advice and tested the polarity of human primary T cells (CD4+, activated and non-activated) and monocytes (CD14+) on osteoblasts (new . None of them were polarized, thus confirming that this property was specific to HSPC.
## Minor:
1. , centrosome shown in white in the image but black in the label.
We corrected this.
2. , no "murine liver-derived mesenchymal stromal cells" despite cited in the text (page 5).
We apologize for the lack of clarity due to the use of abbreviations. AFT and BFC are murine liver-derived stromal cells (see .
3. I've found the terminology of stromal cells and endothelial cells a bit confusing. Are they referring to the same cell types? If so, why not combining these two terms?
We thank the reviewer for pointing at this confusion. The term "stromal cells" was intended to refer to both endothelial cells and osteoblasts at once and remain concise. We made this clearer in the revised text.
## Reviewer #2
This is a nice paper that reports a new phenomena: i.e. that hematopoietic progenitors polarize in response to contact with bone marrow stromal cells. The authors report that this seems to be specific to engagement to CXCR4 receptors. The paper is well written and the observation is interesting in that it extends the idea that cells of the hematopoietic lineage polarize the centrosome in response to signalling receptors. This would be the first report of centrosome polarization in response to a chemokine receptor. What the paper does not do is to explore the mechanism of triggering via this receptor beyond the experiment reported in . Two ways in which they could do so would be :
We thank the reviewer for these positive comments and constructive criticisms which have helped us to improve our study.
1. In NK cells, polarization has been shown to be triggered by integrins and one critical aspect of this has been that the tethering of ICAM seemed to be required for polarization . It would be interesting to know whether CXCR4 engagement requires tethering and whether it activates the integrins-ie could this mechanism be related?
We performed additional immuno-stainings and found that ICAM-1 is enriched in the part of the stromal cells facing the HSPC and that LFA-1 is concentrated in the HSPC protrusion (new so their interaction may be involved in the polarization process. However this interaction is not sufficient since HSPC did not polarize in contact with a surface coated with ICAM-1 only . We added the reference to the work of Gross et al to discuss the difference with NK cells.
## 2.
Do the progenitor cells show signs of intracellular activation? 3. What would be the biological relevance of this polarization? This is a great suggestion. We repeated the experiments of and quantified the occurrence of polarized HSPC in the bone marrow (new . The quantification of the polarized phenotypes of were quantified in 3. The authors conclude that the interaction is similar to an immunological synapse based on sharing some molecules and centrosome polarization. But this doesn't seem plausible. In fact, earlier work showed that this stalk like interaction could be replicated for cytotoxic T cells with surfaces presenting only pMHC (PMID: 15851656), but this was not a highly functional synaptic morphology as the cytotoxic granules weren't recruited towards the target. This study could also be cited to even contrast the stalk like interactions, that can also be generated by TCR signals in the absence of adhesion molecule engagement. So I think on balance, the literature would argue that the stalk like attachments are very distinct from the spreading posture of the T cell forming immunological synapses.
We fully agree with the reviewer and apologize for the misunderstanding. We acknowledged in the text that the exchange of signals, which is required to use the term "synapse", deserves further characterization. We also specifically mentioned the absence of spreading that distinguish the magnupodium from an immunological synapse: "The contact site was restricted to a small area estimated to be around 1-2 µm 2 . However, HSPCs were not observed to spread on osteoblasts, in contrast to a lymphocyte forming immune synapse on a target cell ."
In our revised discussion we made clearer that the magnupodium is distinct from lymphocyte's uropod and immune synapse, but that in term of anchorage, internal architecture and composition it resembles more the latter. Thank you for submitting your revised manuscript entitled "Hematopoietic progenitors polarize in contact with bone marrow stromal cells by engaging CXCR4/SDF1 axis". The paper has now been seen again by two of the original reviewers, both of whom now support acceptance so we would be happy to publish your paper in JCB pending final revisions necessary to meet our formatting guidelines (see details below). **As you will see, reviewer #3 continues to feel that it is important for you to cite the 1992 study that s/he indicated in the previous round of review "both for reporting the mode of adhesion and demonstrating a priori that its not HSC specific". We feel that while adding this citation may not conclusively establish identity between these previous observations and your own, we do think it would be interesting for the community and our readership for you to refer to this paper for purposes of completeness and scholarship. Please be sure to indicate in your final cover letter or rebuttal document how you addressed this point.** To avoid unnecessary delays in the acceptance and publication of your paper, please read the following information carefully.
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Mitochondrial ATP synthase inhibitory factor 1 interacts with the p53–cyclophilin D complex and promotes opening of the permeability transition pore
Edited by Phyllis HansonThe mitochondrial permeability transition pore (PTP) is a Ca 2+ -dependent megachannel that plays an important role in mitochondrial physiology and cell fate. Cyclophilin D (CyPD) is a well-characterized PTP regulator, and its binding to the PTP favors pore opening. It has previously been shown that p53 physically interacts with CyPD and opens the PTP during necrosis. Accumulating studies also suggest that the F-ATP synthase contributes to the regulation and formation of the PTP. F-ATP synthase IF1 (mitochondrial ATP synthase inhibitory factor 1) is a natural inhibitor of F-ATP synthase activity; however, whether IF1 participates in the modulation of PTP opening is basically unknown. Here, we demonstrate using calcium retention capacity assay that IF1 overexpression promotes mitochondrial permeability transition via opening of the PTP. Intriguingly, we show that IF1 can interact with the p53-CyPD complex and facilitate cell death. We also demonstrate that the presence of IF1 is necessary for the formation of p53-CyPD complex. Therefore, we suggest that IF1 regulates the PTP via interaction with the p53-CyPD complex, and that IF1 is necessary for the inducing effect of p53-CyPD complex on PTP opening.
The mitochondrial permeability transition pore (PTP) is a Ca 2+ -dependent megachannel that plays an important role in mitochondrial physiology and cell fate. Cyclophilin D (CyPD) is a well-characterized PTP regulator, and its binding to the PTP favors pore opening. It has previously been shown that p53 physically interacts with CyPD and opens the PTP during necrosis. Accumulating studies also suggest that the F-ATP synthase contributes to the regulation and formation of the PTP. F-ATP synthase IF1 (mitochondrial ATP synthase inhibitory factor 1) is a natural inhibitor of F-ATP synthase activity; however, whether IF1 participates in the modulation of PTP opening is basically unknown. Here, we demonstrate using calcium retention capacity assay that IF1 overexpression promotes mitochondrial permeability transition via opening of the PTP. Intriguingly, we show that IF1 can interact with the p53-CyPD complex and facilitate cell death. We also demonstrate that the presence of IF1 is necessary for the formation of p53-CyPD complex. Therefore, we suggest that IF1 regulates the PTP via interaction with the p53-CyPD complex, and that IF1 is necessary for the inducing effect of p53-CyPD complex on PTP opening.
Mitochondrial calcium overload and oxidative stress may trigger opening of the mitochondrial permeability transition pore (PTP), resulting in the rupture of outer mitochondrial membrane and the depletion of ATP, eventually apoptotic and necrotic cell death [bib_ref] Mitochondria and the permeability transition pore in cancer metabolic reprogramming, Guo [/bib_ref]. Cyclophilin D (CyPD), encoded by PPIF (peptidylprolyl isomerase F) gene, is a most well-characterized regulator of PTP. Binding of CyPD to PTP sensitizes the pore to Ca 2+ , whereas its genetic ablation or detachment from PTP by cyclosporine A (CsA) suppresses the pore opening [bib_ref] Cyclophilin D in mitochondrial pathophysiology, Giorgio [/bib_ref] [bib_ref] Interactions of cyclophilin with the mitochondrial inner membrane and regulation of the..., Nicolli [/bib_ref]. CyPD modulates the activity of F-ATP synthase through its association to the lateral stalk of the enzyme [bib_ref] Dimers of mitochondrial ATP synthase form the permeability transition pore, Giorgio [/bib_ref]. CyPD interacts with oligomycin sensitivity conferral protein (OSCP) of F-ATP synthase and induces conformational changes transferred to F O sector, which could also favor Ca 2+ binding to the catalytic core [bib_ref] OSCP subunit of mitochondrial ATP synthase : Role in regulation of enzyme..., Giorgio [/bib_ref]. The most recent molecular model of PTP formation is that the conformational change induced by Ca 2+ binding to subunit β transmits to e-subunit and c-subunit ring via subunits b and g through OSCP [bib_ref] Ca 2+ binding to F-ATP synthase β subunit triggers the mitochondrial permeability..., Giorgio [/bib_ref] [bib_ref] Arg-8 of yeast subunit e contributes to the stability of F-ATP synthase..., Guo [/bib_ref] [bib_ref] Arginine 107 of yeast ATP synthase subunit g mediates sensitivity of the..., Guo [/bib_ref] [bib_ref] Cryo-EM structure of the entire mammalian F-type ATP synthase, Pinke [/bib_ref].
The tumor suppressor p53, encoded by TP53 gene, is a wellknown transcription factor that exerts diverse biological functions, such as DNA repair, cell cycle, apoptosis, and metabolism [bib_ref] Transcriptional control of human p53-regulated genes, Riley [/bib_ref]. Direct activation of Bax by p53 may induce mitochondrial membrane permeabilization, which participates in apoptotic program [bib_ref] Pharmacologic activation of p53 elicits Bax-dependent apoptosis in the absence of transcription, Chipuk [/bib_ref] [bib_ref] Direct activation of bax by p53 mediates mitochondrial membrane permeabilization and apoptosis, Chipuk [/bib_ref]. OSCP subunit has been identified as a new partner of mitochondrial matrix p53, which promotes assembly of F-ATP synthase and mitochondrial respiration [bib_ref] Mitochondrial p53 mediates a transcription-independent regulation of cell respiration and interacts with..., Bergeaud [/bib_ref]. Oxidative stress induces PTP opening in a p53-dependent manner by its interaction with CyPD [bib_ref] opens the mitochondrial permeability transition pore to trigger necrosis, Vaseva [/bib_ref]. A robust p53-CypD complex is induced by H 2 O 2 treatment, and p53-CypD axis acts as an important contributor to ischemic stroke [bib_ref] opens the mitochondrial permeability transition pore to trigger necrosis, Vaseva [/bib_ref].
F-ATP synthase IF1 (mitochondrial ATP synthase inhibitory factor 1), encoded by ATP5IF1 gene, is a natural inhibitor of F-ATP synthase activity [bib_ref] A naturally occurring inhibitor of mitochondrial adenosine triphosphatase occurring adenosine inhibitor of..., Pullman [/bib_ref]. The oligomerization state of IF1 and its binding to F-ATP synthase depend on the matrix pH [bib_ref] Modulation of the oligomerization state of the bovine F1-ATPase inhibitor protein, IF1,..., Cabezon [/bib_ref] [bib_ref] The regulation of catalysis in ATP synthase, Walker [/bib_ref]. When the proton motive force is present, IF1 releases from F-ATP synthase and ATP synthesis recovers, indicating that IF1 contributes to preventing futile ATP hydrolysis [bib_ref] The ATP synthase: The understood, the uncertain and the unknown, Walker [/bib_ref]. Phosphorylation of human IF1 by PKA abrogates its binding to F-ATP synthase [bib_ref] PKA phosphorylates the ATPase inhibitory factor 1 and inactivates its capacity to..., García-Bermúdez [/bib_ref]. The active dimeric IF1 can bridge the F1-F1 part and promote dimerization of F-ATP synthase [bib_ref] Dimerization of bovine F1-ATPase by binding the inhibitor protein, IF1, Cabezón [/bib_ref] [bib_ref] The inhibitor protein (IF1) promotes dimerization of the mitochondrial F1F0-ATP synthase, García [/bib_ref]. IF1 overexpression favors the formation of F-ATP synthase dimers and inhibits the enzyme activity, accompanied with increased density of mitochondrial cristae, thus, IF1 regulates mitochondrial biogenesis and morphology [bib_ref] Regulation of mitochondrial structure and function by the F1Fo-ATPase inhibitor protein, Campanella [/bib_ref] [bib_ref] The mitochondrial ATPase inhibitory factor 1 triggers a ROS-mediated retrograde prosurvival and..., Formentini [/bib_ref]. IF1 is upregulated in many human tumors and participates in oncogenesis, mediating the metabolic switch to a Warburg phenotype [bib_ref] Up-regulation of the ATPase inhibitory Factor 1 (IF1) of the mitochondrial H+-ATP..., Sánchez-Cenizo [/bib_ref]. IF1 overexpression in colon cancer results in generation of reactive oxygen species (ROS), which triggers a prosurvival and proliferative response [bib_ref] The mitochondrial ATPase inhibitory factor 1 triggers a ROS-mediated retrograde prosurvival and..., Formentini [/bib_ref]. The relative expression level of IF1 varies between different tissues and cell types [bib_ref] Regulation of mitochondrial structure and function by the F1Fo-ATPase inhibitor protein, Campanella [/bib_ref] , which may contribute to heterogeneous phenotypes of tumors in metabolic phenotypes and chemoresistance [bib_ref] Cancer metabolism: A therapeutic perspective, Martinez-Outschoorn [/bib_ref].
Despite the physiological roles of IF1 in regulating metabolic reprogramming and cell fate, the mechanism through which IF1 fulfils these functions is not fully understood.
Whether IF1 plays a role in regulating PTP opening and its molecular mechanism awaits an answer. The present study has addressed the regulatory role of IF1 in PTP opening and cell death via its interaction with p53-CyPD complex.
# Results
## If1 regulates mitochondrial membrane permeabilization
Many tumor tissues are not defective in mitochondrial oxidative phosphorylation (OXPHOS) but rely on oxidative respiration for their proliferation and metastasis [bib_ref] Metabolic reprogramming : A cancer hallmark even warburg did not anticipate, Ward [/bib_ref] [bib_ref] Mitochondrial fusion exploits a therapeutic vulnerability of pancreatic cancer, Yu [/bib_ref]. Pancreatic carcinoma is a high OXPHOS subtype, and it is highly sensitive to an OXPHOS inhibitor phenformin that overcomes chemoresistance [bib_ref] Targeting mitochondrial complex I overcomes chemoresistance in high, Masoud [/bib_ref]. In human pancreatic ductal adenocarcinoma (PDAC) cell line MIA PaCa-2, IF1 expression was notably upregulated [fig_ref] Figure 1: IF1 regulates mitochondrial membrane permeabilization [/fig_ref]. The expression level of p53 in MIA PaCa-2 cells was significantly increased compared with other cancer cell lines like HCT116, HeLa, and HepG2 [fig_ref] Figure 1: IF1 regulates mitochondrial membrane permeabilization [/fig_ref]. The expression level of CyPD in MIA PaCa-2 cells was slightly increased but not significant [fig_ref] Figure 1: IF1 regulates mitochondrial membrane permeabilization [/fig_ref] , A and D). PDAC is a most lethal cancer. The regulatory mechanism and activity of PTP in PDAC is interesting to be known. Overexpression of IF1 caused activation of caspase 3 (Figs. 1, E and F and S1), which was diminished by the treatment of CsA [fig_ref] Figure 1: IF1 regulates mitochondrial membrane permeabilization [/fig_ref]. These observations indicated that IF1 could play a role in modulation of mitochondrial membrane permeabilization.
Overexpression of IF1 sensitizes the PTP to Ca 2+ and promotes cell death Calcium retention capacity (CRC) assay revealed that IF1 overexpression sensitized the pore to Ca 2+ [fig_ref] Figure 2: Overexpression of IF1 sensitizes the PTP to Ca 2+ and promotes cell... [/fig_ref] , A-C), which was prevented by the addition of CsA [fig_ref] Figure 2: Overexpression of IF1 sensitizes the PTP to Ca 2+ and promotes cell... [/fig_ref].
Overexpression of IF1 favored cell death, which was evaluated by ATP assay (Figs. 2F and S2A) and by the observation of cell density and morphology under a microscope (Figs. 2G and S2B). IF1 overexpression inhibited cell growth and promoted cell death probably through induction of PTP opening.
Ablation of IF1 desensitizes the PTP to Ca 2+ and prevents the cell toxicity of t-butyl hydroperoxide IF1 is encoded by the ATPIF1 gene, and ΔATPIF1 mutant was generated to abolish the protein expression of IF1 [fig_ref] Figure 3: Ablation of IF1 desensitizes the PTP to Ca 2+ and prevents the... [/fig_ref]. The role of IF1 in regulation of PTP opening was strengthened by the tests in ΔATPIF1 mutant. Ablation of IF1 strikingly desensitized the pore to Ca 2+ , resulting in a significant increase of CRC [fig_ref] Figure 3: Ablation of IF1 desensitizes the PTP to Ca 2+ and prevents the... [/fig_ref] , B-D). To further confirm the regulatory role of IF1 in PTP opening, the ΔPPIF mutant was generated to genetic disruption of CyPD expression, which is a well-characterized PTP regulator [fig_ref] Figure 3: Ablation of IF1 desensitizes the PTP to Ca 2+ and prevents the... [/fig_ref]. t-Butyl hydroperoxide (tBHP) led to cell death in a mitochondrial permeability transition-dependent manner [bib_ref] Mitochondria permeability transition-dependent tert-butyl hydroperoxide-induced apoptosis in hepatoma HepG2 cells, Piret [/bib_ref]. About 20 micromolar of tBHP induced about 90% of MIA PaCa-2 cells dead after 24 h, and knockout of either IF1 or PPIF was able to prevent the cell toxicity of tBHP [fig_ref] Figure 3: Ablation of IF1 desensitizes the PTP to Ca 2+ and prevents the... [/fig_ref]. These observations suggested that knockout of IF1 inhibited PTP opening and prevented the cell death induced by oxidative stress.
## If1 interacts with p53-cypd complex
The molecular mechanism through which IF1 regulates PTP was investigated. IF1 binds to a α DP -β DP catalytic interface of F-ATP synthase and also contacts subunit γ (33-35). Binding of Ca 2+ to the catalytic core of F-ATP synthase causes a conformational change that transmits to the inner membrane subunits through the lateral stalk via OSCP, which triggers PTP formation [bib_ref] Calcium and regulation of the mitochondrial permeability transition, Giorgio [/bib_ref]. OSCP subunit is one of the major interacting proteins with CyPD (7), and p53-CypD complex is required for PTP opening under oxidative stress [bib_ref] opens the mitochondrial permeability transition pore to trigger necrosis, Vaseva [/bib_ref]. The possible interaction of IF1 with p53-CypD complex and its role in regulation of PTP were explored.
The mechanism through which IF1 regulates the permeability transition was investigated in human embryonic kidney 293T (HEK293T) cells. IF1 interacted with subunit α within mitochondria, but the interaction of exogenous OSCP with subunit α was not able to be detected [fig_ref] Figure 5: IF1 interacts with p53-CyPD complex [/fig_ref] , indicating that the conformational change induced by IF1 binding was transferred to PTP probably through an alternative pathway. IF1 and CyPD are two mitochondrial-specific proteins, and p53 is partly located in mitochondrial matrix under physiological condition [fig_ref] Figure 5: IF1 interacts with p53-CyPD complex [/fig_ref]. IF1 was able to interact with CyPD and OSCP [fig_ref] Figure 5: IF1 interacts with p53-CyPD complex [/fig_ref]. Disruption of either ATP1F1 or PPIF prevented the stable expression of p53 [fig_ref] Figure 5: IF1 interacts with p53-CyPD complex [/fig_ref]. The interaction of IF1 with p53 was able to be detected [fig_ref] Figure 5: IF1 interacts with p53-CyPD complex [/fig_ref]. The interaction between IF1 and p53-CyPD complex could not be enhanced by tBHP [fig_ref] Figure 4: Ablation of CyPD blocks the inducing effect of IF1 overexpression on PTP [/fig_ref] , indicating that IF1 favored PTP opening through a mechanism different from that of tBHP. These results suggested that IF1 modulated PTP probably through p53-CypD axis.
## If1 combined with p53-cypd complex facilitates cell death
Overexpression of IF1 sensitized PTP to Ca 2+ in MIA PaCa-2 [fig_ref] Figure 2: Overexpression of IF1 sensitizes the PTP to Ca 2+ and promotes cell... [/fig_ref] , A-E) and HEK293T cells , A-C). IF1 was able to interact with p53-CyPD complex; therefore, p53 and CyPD were also overexpressed to investigate their effects on mitochondrial CRC and cell death. Either overexpression of p53 or CyPD alone did not influence the CRC unless their combination was used, suggesting that p53 or CyPD was not sufficient for the pore opening . IF1 alone or its combination with p53 and CyPD favors the pore opening . Unexpectedly, IF1 and p53-CyPD complex did not exhibit synergistic effect on the CRC . The presence of . Error bars indicated the mean ± SD of five independent experiments. *p < 0.05, ns, Student's t test. E, CRC/CRC 0 ratio of digitonin-permeabilized MIA PaCa-2 cells transfected with ATPIF1 normalized to those transfected with EV. CRC values of permeabilized MIA PaCa-2 cells transfected with EV were set as one unit. Black columns and gray columns represent CRC/CRC 0 ratio in the absence of CsA or in the presence of CsA, respectively. Error bars indicated the mean ± SD of five independent experiments. ***p < 0.001, ns, two-way ANOVA with Bonferroni post hoc test. F, the effect of IF1 overexpression on cell viability in MIA PaCa-2 cells revealed by ATP assay. Error bars indicated the mean ± SD of six independent experiments. ***p < 0.001 versus EV, Student's t test. G, the images of MIA PaCa-2 cells transfected with EV or ATPIF1 for 24 h (the scale bar represents 100 μm). The figures are representative of at least three independent experiments. CRC, calcium retention capacity; CsA, cyclosporine A; EV, empty vector; IF1, mitochondrial ATP synthase inhibitory factor 1; ns, not significant; PTP, the mitochondrial permeability transition pore; WB, Western blotting. independent experiments. F, histograms represent the relative densitometry values of cleaved caspase 3 normalized to caspase 3. Error bars indicated the mean ± SD of three independent experiments. *p < 0.05, one-way ANOVA with Bonferroni post hoc test. G and H, MIA PaCa-2 cells were transfected with EV or ATPIF1 and treated with 2 μM CsA for 24 h. Cell lysates were analyzed by WB. G, representative blots of three independent experiments. H, histograms represent the relative densitometry values of cleaved caspase 3 normalized to caspase 3. Error bars indicated the mean ± SD of three independent experiments. *p < 0.05, ***p < 0.001, one-way ANOVA with Bonferroni post hoc test. CsA, cyclosporine A; CyPD, cyclophilin D; HA, hemagglutinin; HEK293T, human embryonic kidney 293T cell line; IF1, mitochondrial ATP synthase inhibitory factor 1; MEF, mouse embryonic fibroblast. IF1 regulates PTP via p53-CyPD CsA prevented the effects of overexpression of IF1 or p53-CyPD complex on PTP . These observations indicated that CyPD was not a sufficient factor for the pore opening but necessary for IF1-induced PTP formation. Binding of IF1 to F-ATP synthase induced a conformational change that transmitted to the inner membrane subunits probably through the lateral stalk via CyPD. IF1 inhibited ATP hydrolysis but not ATP synthesis of the enzyme [bib_ref] An inhibitor of the F1 subunit of ATP synthase (IF1) modulates the..., Burwick [/bib_ref] , which allowed ATP assay to detect the effect of IF1 overexpression on cell viability. Overexpression of IF1 slightly promoted cell death, which was dramatically facilitated by its combination with p53 and CyPD . IF1 and p53-CyPD complex showed synergistic effect on cell death . The effects of IF1 and p53-CyPD complex on cell death appeared to be contradictory to those on CRC. This discrepancy could be due to the possibility that the binding of Ca 2+ to the pore weakened the impact of p53-CyPD overexpression on the conformational change of PTP.
## P53 interacts with cypd in an if1-dependent manner
Whether the binding of IF1 to F-ATP synthase modulates the inducing effect of p53-CyPD complex on PTP was then investigated. In MIA PaCa-2 cells, overexpression of p53-CyPD complex dramatically sensitized the pore to Ca 2+ , which was completely abolished by the knockout of IF1. Cell death was facilitated by overexpression of p53-CyPD complex in WT MIA PaCa-2 cells, which was disappeared in ΔATPIF1 mutant. Consistent with the previous study [bib_ref] opens the mitochondrial permeability transition pore to trigger necrosis, Vaseva [/bib_ref] , the interaction of p53 with CyPD was able to be detected in WT MIA PaCa-2 cells. Remarkably, knockout of IF1 prevented the interaction of p53 with CyPD. These observations suggested that IF1 was necessary for the p53-CyPD complex formation, probably because the binding of IF1 to F-ATP synthase caused a conformational change that favored the interaction of p53 with CyPD.
# Discussion
This work reports a new role of IF1 in modulation of PTP. IF1, an intrinsic inhibitor of F-ATP synthase, inhibits ATP hydrolysis and has no direct effect on ATP synthesis in the presence of a proton motive force [bib_ref] A naturally occurring inhibitor of mitochondrial adenosine triphosphatase occurring adenosine inhibitor of..., Pullman [/bib_ref]. Recent studies demonstrate that overexpressing IF1 inhibits both ATP hydrolysis and synthesis activities of F-ATP synthase [bib_ref] PKA phosphorylates the ATPase inhibitory factor 1 and inactivates its capacity to..., García-Bermúdez [/bib_ref]. IF1 limits OXPHOS and thus promotes glycolysis, mediating metabolic switch in cancer cells [bib_ref] Up-regulation of the ATPase inhibitory Factor 1 (IF1) of the mitochondrial H+-ATP..., Sánchez-Cenizo [/bib_ref]. Besides regulating the activities of the enzyme, IF1 exerts additional roles in mitochondrial physiology [bib_ref] The mitochondrial permeability transition pore: Channel formation by F-ATP synthase, integration in..., Bernardi [/bib_ref]. Inhibition of F-ATP synthase activity by IF1 increases mitochondrial membrane potential (ΔΨ m ) because of the inhibition of H + translocation into matrix [bib_ref] The mitochondrial ATPase inhibitory factor 1 triggers a ROS-mediated retrograde prosurvival and..., Formentini [/bib_ref] [bib_ref] Up-regulation of the ATPase inhibitory Factor 1 (IF1) of the mitochondrial H+-ATP..., Sánchez-Cenizo [/bib_ref]. Mitochondrial hyperpolarization triggers generation of ROS, which mediates cellular proliferation and survival [bib_ref] The mitochondrial ATPase inhibitory factor 1 triggers a ROS-mediated retrograde prosurvival and..., Formentini [/bib_ref] [bib_ref] The mitochondrial F0F1-ATPase proton pump is required for function of the proapoptotic..., Matsuyama [/bib_ref] [bib_ref] Efficient execution of cell death in non-glycolytic cells requires the generation of..., Santamaría [/bib_ref]. Relative IF1 expression varies between different tissues and cell types [bib_ref] Regulation of mitochondrial structure and function by the F1Fo-ATPase inhibitor protein, Campanella [/bib_ref] [fig_ref] Figure 1: IF1 regulates mitochondrial membrane permeabilization [/fig_ref].
Overexpression of IF1 in HeLa cells instead downregulates the ΔΨ m [bib_ref] Regulation of mitochondrial structure and function by the F1Fo-ATPase inhibitor protein, Campanella [/bib_ref]. MIA PaCa-2 cell lines displayed high basal expression of IF1 [fig_ref] Figure 1: IF1 regulates mitochondrial membrane permeabilization [/fig_ref]. PDAC is the most lethal cancer, which is usually diagnosed at an advanced stage and is refractory to all currently available treatments [bib_ref] Pancreatic adenocarcinoma, Meslar [/bib_ref]. PDAC cancer cell line MIA PaCa-2 was used to assess the influence of IF1 overexpression on mitochondrial permeability transition and whether the decreased ΔΨ m was due to the opening of PTP. IF1 binding to F-ATP synthase not only is regulated by the energetic state of mitochondria [bib_ref] How the regulatory protein, IF1, inhibits F1-ATPase from bovine mitochondria, Gledhill [/bib_ref] but also depends on the molar ratio because the mass-action ratio controls the interaction between proteins [bib_ref] Up-regulation of the ATPase inhibitory Factor 1 (IF1) of the mitochondrial H+-ATP..., Sánchez-Cenizo [/bib_ref]. Overexpression of IF1 triggered the activation of caspase 3 [fig_ref] Figure 1: IF1 regulates mitochondrial membrane permeabilization [/fig_ref] , which was suppressed by the well-established PTP inhibitor CsA [fig_ref] Figure 1: IF1 regulates mitochondrial membrane permeabilization [/fig_ref]. Overexpression of IF1 sensitized the PTP opening to Ca 2+ , which was abrogated by CsA [fig_ref] Figure 2: Overexpression of IF1 sensitizes the PTP to Ca 2+ and promotes cell... [/fig_ref] , A-E) or genetic ablation of CyPD [fig_ref] Figure 4: Ablation of CyPD blocks the inducing effect of IF1 overexpression on PTP [/fig_ref]. Knockout of CyPD abolished the inducing effect of cell death by IF1 overexpression (Figs. 2, F and G and 4A). Genetic ablation of IF1 desensitized the PTP opening to Ca 2+ [fig_ref] Figure 3: Ablation of IF1 desensitizes the PTP to Ca 2+ and prevents the... [/fig_ref] and prevented cell death induced by tBHP, which displayed similar influence to that of CyPD [fig_ref] Figure 3: Ablation of IF1 desensitizes the PTP to Ca 2+ and prevents the... [/fig_ref]. These data suggested that promoted binding of IF1 to F-ATP synthase triggered mitochondrial permeability transition. Transient opening of the PTP participates in Ca 2+ /ROS homeostasis and chemoresistance (1), which may favor the malignance of PDAC with high expression of IF1. Next, the molecular mechanism through which IF1 favored PTP opening was then investigated.
IF1 binds to F-ATP synthase in a matrix pH-dependent manner [bib_ref] Modulation of the oligomerization state of the bovine F1-ATPase inhibitor protein, IF1,..., Cabezon [/bib_ref] [bib_ref] The regulation of catalysis in ATP synthase, Walker [/bib_ref]. IF1 overexpression promotes oligomerization of F-ATP synthase and the formation of mitochondrial cristae, thus, IF1 plays a role in mitochondrial biogenesis and morphology [bib_ref] Regulation of mitochondrial structure and function by the F1Fo-ATPase inhibitor protein, Campanella [/bib_ref] [bib_ref] The mitochondrial ATPase inhibitory factor 1 triggers a ROS-mediated retrograde prosurvival and..., Formentini [/bib_ref]. Overwhelming evidences support the hypothesis that F-ATP synthase contributes to the regulation and formation of PTP through electrophysiological analysis of purified enzymes [bib_ref] A mitochondrial megachannel resides in monomeric F1FO ATP synthase, Mnatsakanyan [/bib_ref] [bib_ref] Purified F-ATP synthase forms a Ca2+-dependent high-conductance channel matching the mitochondrial permeability..., Urbani [/bib_ref] and by site-directed mutagenesis [bib_ref] Arg-8 of yeast subunit e contributes to the stability of F-ATP synthase..., Guo [/bib_ref] [bib_ref] Arginine 107 of yeast ATP synthase subunit g mediates sensitivity of the..., Guo [/bib_ref] [bib_ref] An uncoupling channel within the c-subunit ring of the F1FO ATP synthase..., Alavian [/bib_ref] [bib_ref] The unique histidine in OSCP subunit of F-ATP synthase mediates inhibition of..., Antoniel [/bib_ref] [bib_ref] The unique cysteine of F-ATP synthase OSCP subunit participates in modulation of..., Carraro [/bib_ref]. The hypothesis that PTP forms from F-ATP synthase has been questioned by the studies based on the disruption of F-ATP synthase subunits [bib_ref] Persistence of the mitochondrial permeability transition in the absence of subunit c..., He [/bib_ref] [bib_ref] Permeability transition in human mitochondria persists in the absence of peripheral stalk..., He [/bib_ref] [bib_ref] Persistence of the permeability transition pore in human mitochondria devoid of an..., Carroll [/bib_ref] [bib_ref] Reply to BERNARDI : The mitochondrial permeability transition pore and the ATP..., Walker [/bib_ref]. In human mitochondria devoid of an assembled ATP synthase, the persistent observations of CsA-sensitive Ca 2+ release and mitochondrial swelling in potassium chloride (KCl)-based buffer may be due to a distinct permeability pathway, which is provided by the adenine nucleotide translocator (ANT) [bib_ref] ATP synthase C-subunit-deficient mitochondria have a small cyclosporine A-sensitive channel, but lack..., Neginskaya [/bib_ref]. Crosslinking and proteolysis experiments reveal that the central segment of IF1 binds to αβ subunits of F 1 sector in a pH-dependent process and inhibits F-ATP synthase activity [bib_ref] Inhibitory and anchoring domains in the ATPase inhibitor protein IF 1 of..., Zanotti [/bib_ref]. Interestingly, the C-terminal region of IF1 simultaneously binds to the OSCP subunit of F O sector in a pH-independent process [bib_ref] Inhibitory and anchoring domains in the ATPase inhibitor protein IF 1 of..., Zanotti [/bib_ref]. This binding keeps IF1 anchored to F-ATP synthase both at acidic pH and alkaline pH [bib_ref] Inhibitory and anchoring domains in the ATPase inhibitor protein IF 1 of..., Zanotti [/bib_ref]. OSCP subunit acts as a flexible hinge by connection F 1 and F O , indicating that revealed by ATP assay after 24 h of treatment. Viability is presented as a percentage (%) of the control value without the treatment of tBHP. Error bars indicated the mean ± SD of at least two independent experiments. *p < 0.05 versus WT, ***p < 0.001 versus WT, two-way ANOVA with Bonferroni post hoc test. CRC, calcium retention capacity; CyPD, cyclophilin D; IF1, mitochondrial ATP synthase inhibitory factor 1; PTP, the mitochondrial permeability transition pore; tBHP, t-butyl hydroperoxide. OSCP is a hub of metabolic control [bib_ref] Rotary substates of mitochondrial ATP synthase reveal the basis of flexible F1-Fo..., Murphy [/bib_ref]. Binding of CyPD and the immunomodulator Bz-423 to OSCP subunit sensitizes the pore to Ca 2+ and inhibits F-ATP synthase activity [bib_ref] Identification and validation of the mitochondrial F1F0-ATPase as the molecular target of..., Johnson [/bib_ref]. The tumor suppressor p53 interacts with OSCP subunit via p53-CypD axis, which plays an important role in mitochondrial physiology and tumor suppression activity [bib_ref] Mitochondrial p53 mediates a transcription-independent regulation of cell respiration and interacts with..., Bergeaud [/bib_ref] [bib_ref] opens the mitochondrial permeability transition pore to trigger necrosis, Vaseva [/bib_ref]. IF1 appears to extend beyond what envisaged in the literature, and to know better about this fascinating little protein await more studies.
## If1 regulates ptp via p53-cypd
In this study, IF1 was found to interact with p53 and CyPD, as well as OSCP subunit, and either ablation of IF1 or ablation of CyPD affected the stable expression of p53 [fig_ref] Figure 5: IF1 interacts with p53-CyPD complex [/fig_ref]. Overexpression of IF1 and p53-CyPD complex sensitized the pore opening to Ca 2+ , which was inhibited by the addition of CsA . In addition, IF1 combined with p53-CyPD complex promoted cell death . These observations indicated that IF1 interacted with p53-CyPD complex resulting in a conformational change that transmitted to PTP via OSCP subunit. In the last, p53 appeared to interact with CyPD in an IF1-dependent manner. treated mitochondria with the membrane-permeable bifunctional reagent dimethyl 3,3-dithiobis-propionimidate prior to immunoprecipitation (IP) with anti-complex V antibody and found that CyPD associated to the lateral stalk of F-ATP synthase but not interacted with IF1 and ANT. These different observations might be due to the different techniques used. Some interactions could be missing because crosslinks with dimethyl 3,3-dithiobis-propionimidate is cleavable by DTT. Indeed, the interaction between CyPD and ANT exists, and ANT forms a CsA-sensitive channel, which acts as a distinct permeability pathway [bib_ref] ATP synthase C-subunit-deficient mitochondria have a small cyclosporine A-sensitive channel, but lack..., Neginskaya [/bib_ref]. In this work, overexpression of IF1 probably further strengthened its interaction with CyPD. B, HEK293T cells were cultured for preparation of isolated mitochondria (mito) and cytosolic fraction (cyto), which were lysed for WB analysis. The blots are representative of three independent experiments. C, HEK293T cells were transfected with EV or plasmids carrying ATPIF1 extracted from two different Escherichia coli colonies for 24 h. Cell lysates were prepared for co-IP and analyzed by WB. The blots are representative of three independent experiments. D, HCT116 cells were transduced with lentiCRISPRv2 construct containing scrambled guide RNA (Scr) or two different single-guide RNAs targeting ATPIF1 or PPIF (sg1 or sg2) and selected by 2 μg/ml puromycin for about 7 days. Cell lysates were prepared to detect the expression level of p53 by WB analysis. The blots are representative of three independent experiments. E, HEK293T cells were transfected with EV or plasmids carrying ATP5O or ATPIF1 for 24 h. Cell lysates were prepared for co-IP and analyzed by WB. The blots are representative of at least three independent experiments. CyPD, cyclophilin D; EV, empty vector; HEK293T, human embryonic kidney 293T cell line; IF1, mitochondrial ATP synthase inhibitory factor 1; IP, immunoprecipitation; OSCP, oligomycin sensitivity conferral protein; WB, Western blotting. IF1 regulates PTP via p53-CyPD . IF1 combined with p53-CyPD complex facilitates cell death. A, HEK293T cells were transfected with plasmids carrying the indicated genes for 24 h. Cell lysates were analyzed by WB. The blots are representative of at least three independent experiments. B, representative CRC traces of digitoninpermeabilized HEK293T cells transfected with EV (black) or ATPIF1 (red) or three plasmids carrying different genes ATPIF1, TP53, and PPIF (ATPIF1 + TP53 + PPIF) for 24 h. C and D, CRC/CRC 0 ratio of digitonin-permeabilized HEK293T cells transfected with plasmids carrying the indicated genes normalized to those transfected with EV in the absence of CsA (C) or in the presence of CsA (D). CRC values of permeabilized HEK293T cells transfected with EV were set as one unit. Error bars indicated the mean ± SD of at least two independent experiments. *p < 0.05 versus EV, **p < 0.01 versus EV, ***p < 0.001 versus EV, and ****p < 0.0001 versus EV, Student's t test. E, the effect of overexpression of indicated genes on cell viability in HEK293T cells revealed by ATP assay. Error bars indicated the mean ± SD of at least two independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA with Bonferroni post hoc test.
IF1 is known to participate in the Warburg effect of cancer cells [bib_ref] Up-regulation of the ATPase inhibitory Factor 1 (IF1) of the mitochondrial H+-ATP..., Sánchez-Cenizo [/bib_ref]. This work identifies that IF1 played a role in modulation of PTP opening, which provides a new clue that PTP may be involved in regulating metabolic switch, cell fate, and chemoresistance in the development of lethal cancer (1).
## Experimental procedures
Cell culture HEK293T, MIA PaCa-2, HeLa, and HCT116 cells (Cell Bank, Chinese Academy of Sciences) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with fetal bovine serum (10% v/v) and 100 mg/ml penicillin and streptomycin. All the mentioned cell lines were cultured at 37 C in a humidified atmosphere containing 5% CO 2 .
## Plasmid dna transfection
A polyethylenimine (PEI)-mediated transfection method was used in this work. For each well of cells seeded in a 6-well plate, 2 μg plasmids were diluted in 200 μl free DMEM, then 6 μl of PEI was added to the aforementioned solution, and mixed well. The mixture was incubated at room temperature for 15 min and added to each well. For cells cultured in a 10 cm dish, 10 μg plasmids were diluted in 1 ml free DMEM, then 30 μl PEI was added to the aforementioned solution, and mixed well. The mixture was incubated at room temperature CRC, calcium retention capacity; CsA, cyclosporine A; CyPD, cyclophilin D; EV, empty vector; HEK293T, human embryonic kidney 293T cell line; IF1, mitochondrial ATP synthase inhibitory factor 1; WB, Western blotting. . p53 interacts with CyPD in an IF1-dependent manner. A, representative CRC traces of digitonin-permeabilized WT (black) and ΔATPIF1 (red) MIA PaCa-2 cells transfected with C terminus HA-tagged plasmids carrying indicated genes for 24 h. B, CRC/CRC 0 ratio of digitonin-permeabilized WT (black column) and ΔATPIF1 (red column) MIA PaCa-2 cells transfected with plasmids carrying the indicated genes normalized to those transfected with EV. CRC values of permeabilized WT (black) and ΔATPIF1 (red) MIA PaCa-2 cells transfected with EV were set as one unit, respectively. Error bars indicated the mean ± SD of five independent experiments. *p < 0.05, ns, two-way ANOVA with Bonferroni post hoc test. C, the effects of p53-CyPD complex overexpression on cell viability in WT and ΔATPIF1 MIA PaCa-2 cells revealed by ATP assay. Error bars indicated the mean ± SD of six independent experiments. **p < 0.01, ns, two-way ANOVA with Bonferroni post hoc test. D, WT and ΔATPIF1 MIA PaCa-2 cells were nontransfected or transfected with EV or with plasmids carrying ATPIF1 or PPIF or TP53 for 24 h. Protein was extracted for co-IP and WB. The blots are representative of three independent experiments. CRC, calcium retention capacity; CyPD, cyclophilin D; EV, empty vector; HA, hemagglutinin; IF1, mitochondrial ATP synthase inhibitory factor 1; IP, immunoprecipitation; ns, not significant; WB, Western blotting.
for 15 min and added to 10 cm dish. Proteins were extracted, or cells were permeabilized 24 h after the transfection.
## Disruption of ppif and atpif1 in mia paca-2 cells
LentiCRISPR methods was used to disrupt PPIF (encoding CyPD) and ATPIF1 (encoding IF1) genes in MIA PaCa-2 cell line. The specific single-guide RNA (sgRNA) was constructed into a lentiviral expression vector lentiCRISPR v2. The specific sgRNA oligos were designed at the website https://www. benchling.com/crispr/. The target sequences are as follows: PPIF sgRNA-1: 5 0 -CACCGTGACGTCGCGCGCGCCCA GA-3' (forward), 3 0 -AAACTCTGGGCGCGCGCGACGTC AC-5' (reverse); PPIF sgRNA-2: 5 0 -CACCGCGGGAACCCGC TCGTGTACC-3' (forward), 3 0 -AAACGGTACACGAGCGGG TTCCCGC-5' (reverse); PPIF sgRNA-3: 5 0 -CACCGGTACAC GAGCGGGTTCCCGG-3' (forward), 3 0 -AAACCCGGGAA CCCGCTCGTGTACC-5' (reverse); ATPIF1 sgRNA-1: 5 0 -CA CCGCCCGACCGTTGCCACGTATA-3' (forward), 3 0 -AAA CTATACGTGGCAACGGTCGGGC-5' (reverse); ATPIF1 sgRNA-2: 5 0 -CACCGCGCGCCCCGGTCGACATTCT-3' (forward), 3 0 -AAACAGAATGTCGACCGGGGCGCGC-5' (reverse); and ATPIF1 sgRNA-3: 5 0 -CACCGAAAGATGGG ATCCGCCCGCG-3' (forward), 3 0 -AAACCGCGGGCGGAT CCCATCTTTC-5' (reverse). Each pair of oligos was annealed in a thermocycler (37 C for 30 min and 95 C for 5 min and then ramp down to 25 C at 5 C/min). The annealed oligos were cloned into the linearized lentiCRISPR v2 by BsmBI with a ligation kit (Ligation high version 2; Toyobo). For lentivirus production, psPAX2, pMD2.G, and plasmid DNA were diluted with free DMEM at 2:1:6 ratio and transfected to 50% confluence HEK293T cell with PEI-mediated transfection method. The culture medium containing lentivirus was collected and filtered with 0.45 μm filters. The lentivirus suspension was mixed with MIA PaCa-2 cell suspension supplemented with 10 μg/ml polybrene. The complete DMEM was refreshed after 24 h. MIA PaCa-2 cells were cultured for another 24 h. The selection was sustained with 2 μg/ml puromycin until the cells without lentivirus transduction were completely dead. Single cell was seeded into 96-well plate by limiting dilution method and cultured for 10 to 14 days to allow single cell-derived colony formation. Immunoblotting was used to validate the disruption of target genes.
## Generation of hemagglutinin-tagged plasmids carrying target genes
Complementary DNA clones of ATPIF1, ATP5O, PPIF, and TP53 containing XhoI and EcoRI restriction sites were generated by a PCR-based method using the whole complementary DNA of HEK293T as template. The primers used for PCR are as follows: for ATPIF1: 5 0 -ATGCGAATTCATGGC AGTGACGGCGTTGGC-3' (forward), 3 0 -ATGCCTCGAGAT CATCATGTTTTAGCATTT-5' (reverse); for ATP5O: 5 0 -AA GGGATCCGGAATTCATGGCTGCCCCAGCAGTG-3' (forward), 3 0 -TAGATGCATGCTCGAGGACAATCTCCCGCAT AGCCCTG-5' (reverse); for PPIF: 5 0 -CAGTGTGCTGGAA TTCATGCTGGCGCTGCGCTGC-3' (forward), 3 0 -CGTA GGGGTACTCGAGGCTCAACTGGCCACAGTCTGTG-5' (reverse); and for TP53: 5 0 -CAGTGTGCTGGAATTATGG AGGAGCCGCAGTCAG-3' (forward), 3 0 -CGTAGGGGTA CTCGAGTCTGAGTCAGGCCCTTCTGTC-5' (reverse). The PCR products were cloned into a C terminus hemagglutinin (HA)-tagged pCDNA3.1 vector linearized by XhoI and EcoRI restriction enzymes using a ligation high kit (Toyobo). The insertion of target gene was confirmed using DNA sequencing.
Preparation of isolated mitochondria from HEK293T cells HEK293T cells were collected and washed once with cold Dulbecco's PBS by centrifugation at 600g for 5 min. The cell pellet was resuspended with several milliliters of mitochondrial isolation buffer containing 250 mM sucrose, 10 mM Tris-HCl, 0.1 mM EGTA, and pH 7.4. The homogenate suspension was centrifuged at 600g for 10 min. The supernatant was collected and centrifuged at 12,000g for 10 min. The pellet was resuspended in 1 ml isolation buffer and centrifuged at 12,000g for 10 min. The mitochondrial pellet was resuspended in 100 to 200 μl isolation buffer. Bicinchoninic acid assay was used to determine mitochondrial protein concentration.
## Cell permeabilization
Human cells were harvested by a centrifugation at 600g for 5 min. Cells were resuspended and washed with a KCl-based buffer containing 130 mM KCl, 10 mM Mops-Tris, 1 mM Pi, 0.1 mM EGTA, and pH 7.4. The cell pellet was resuspended in a KCl-based buffer containing 130 mM KCl, 10 mM Mops-Tris, 1 mM Pi, 1 mM EGTA, and pH 7.4 supplemented with 0.1 mM digitonin at a density of 2 × 10 7 cells × ml −1 . The incubation with digitonin took about 10 min. The cell permeabilization by digitonin was terminated by addition of 15 ml of a KCl-based buffer containing 130 mM KCl, 10 mM Mops-Tris, 1 mM Pi, 0.1 mM EGTA, and pH 7.4. Cell pellet was collected after a centrifugation at 600g for 5 min and washed once with 5 ml of the same KCl buffer by centrifugation at 600g for 5 min. The cell pellet was resuspended in a sucrosebased buffer (250 mM sucrose, 10 mM Tris-Mops, 10 μM EGTA, pH 7.4) for CRC assay.
## Ip and western blotting
Cells were lysed in co-IP lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, 10% glycerophosphate, and a cocktail of proteinase inhibitors. After incubation on ice for 30 min, cell lysate was harvested by centrifugation at 12,000g for 20 min at 4 C. The cleared cell lysate was incubated with anti-HA agarose beads (Abmart) at 4 C overnight. The beads were washed at least three times in co-IP lysis buffer and boiled in 2× loading buffer for 10 min. Proteins were separated by SDS-PAGE gel electrophoreses and transferred onto nitrocellulose membranes (Millipore). The membranes were blocked with 5% (w/v) nonfat dry milk and incubated with the corresponding primary IF1 regulates PTP via p53-CyPD antibody at 4 C overnight. The immunoblot bands were detected with the Odyssey system (LI-COR Biosciences).
## Mitochondrial ca 2+ retention capacity
Human permeabilized cells were suspended in a sucrosebased assay buffer containing 250 mM sucrose, 10 mM Tris-Mops, 10 μM EGTA, 1 mM Pi, 0.5 μM Calcium green-5N, 5 mM succinate, 2 μM rotenone, and pH 7.4 at a cell density of 1 × 10 7 cells × ml −1 . The cell suspension was added to a black 96-well plate (0.2 ml per well). The concentration of external Ca 2+ was monitored by Calcium Green-5N fluorescence (excitation/emission: 485 nm/538 nm) with a Tecan microplate reader.
## Cell viability assay
Cells were seeded in 96-well plates (100 μl/well) at a cell density of 1 × 10 5 cells × ml −1 1 day before treatment. After 24 h, different doses of tBHP were added as indicated in the figure legends. For overexpression, the PEI-mediated transfection method was used. After 24 h, 10 μl of CellTiter-Glo 2.0 assay (Promega) was added to each well. The plates were incubated at room temperature for 10 min. Luminescence was recorded with a Tecan microplate reader.
## Statistics
All the data were presented as mean ± SD of at least three independent experiments or as representative blots and traces. p Values indicated in the figures are calculated with GraphPad (GraphPad Software, Inc). One-way ANOVA was used to analyze the difference between more than two groups. Twoway ANOVA was used to determine the interaction between two independent variables. *p < 0.05 was considered statistically significant.
## Data availability
All data are contained within the article and supporting information.
Supporting information-This article contains supporting information.
[fig] Figure 1: IF1 regulates mitochondrial membrane permeabilization. A and B, protein extracts of HEK293T, HCT116, CT26, HT29, HeLa, HepG2, Capan-1, PATU8988T, MIA PaCa-2, U937, and MEF cells were analyzed by Western blotting (WB). A, representative blots of five independent experiments. B, histograms represent the relative densitometry values of IF1 normalized to β-actin. Error bars indicated the mean ± SD of five independent experiments. **p < 0.01, ***p < 0.001 versus MIA PaCa-2, one-way ANOVA with Bonferroni post hoc test. C, histograms represent the relative densitometry values of p53 normalized to β-actin. Error bars indicated the mean ± SD of at least five independent experiments. *p < 0.05 versus MIA PaCa-2, one-way ANOVA with Bonferroni post hoc test. D, histograms represent the relative densitometry values of CyPD normalized to β-actin. Error bars indicated the mean ± SD of at least four independent experiments. E and F, MIA PaCa-2 cells were transfected with pcDNA3.1+/C-HA empty vector (EV) or C terminus HA-tagged ATPIF1 plasmids (ATPIF1) extracted from two different Escherichia coli colonies for 24 h. Cell lysates were analyzed by WB. E, representative blots of three [/fig]
[fig] Figure 2: Overexpression of IF1 sensitizes the PTP to Ca 2+ and promotes cell death. A, MIA PaCa-2 cells were transfected with EV or ATPIF1 for 24 h. Cell lysates were analyzed by WB. The blots are representative of at least three independent experiments. B, representative CRC traces of digitoninpermeabilized MIA PaCa-2 cells transfected with EV (black) or ATPIF1 (red) for 24 h. C and D, CRC values of digitonin-permeabilized MIA PaCa-2 cells after transfection of EV (black column) or ATPIF1 (red column) in the absence of CsA (C) or in the presence of CsA (D) [/fig]
[fig] Figure 3: Ablation of IF1 desensitizes the PTP to Ca 2+ and prevents the cell toxicity of tBHP. A, expression of IF1 in MIA PaCa-2 clonal cells after disruption of ATPIF1 gene (encoding IF1) using CRISPR/Cas9 technique. The blots are representative of at least three independent experiments. B, representative CRC traces of digitonin-permeabilized wild-type (WT, black) and ΔATPIF1 (red) MIA PaCa-2 cells. C, CRC values of digitonin-permeabilized WT (black column) and ΔATPIF1 (red column) MIA PaCa-2 cells. Error bars indicated the mean ± SD of seven independent experiments. **p < 0.01 versus WT, Student's t test. D, CRC/CRC 0 ratio of digitonin-permeabilized ΔATPIF1 MIA PaCa-2 cells normalized to WT. CRC values of digitonin-permeabilized WT MIA PaCa-2 cells were set as one unit. Error bars indicated the mean ± SD of seven independent experiments. ***p < 0.001 versus WT, Student's t test. E, expression of CyPD in MIA PaCa-2 clonal cells after disruption of PPIF gene using CRISPR/Cas9 technique. The blots are representative of at least three independent experiments. F, the effect of tBHP on cell viability in WT (black column), ΔATPIF1 (red column), and ΔPPIF (blue column) MIA PaCa-2 cells [/fig]
[fig] Figure 4: Ablation of CyPD blocks the inducing effect of IF1 overexpression on PTP. A, the effect of IF1 overexpression on cell viability in ΔPPIF MIA PaCa-2 cells revealed by ATP assay. Error bars indicated the mean ± SD of six independent experiments. ns, Student's t test. B, representative CRC traces of digitonin-permeabilized WT MIA PaCa-2 cells transfected with EV (black) or ATPIF1 (red) for 24 h. C, CRC/CRC 0 ratio of digitonin-permeabilized WT MIA PaCa-2 cells transfected with ATPIF1 normalized to those transfected with EV. CRC values of digitonin-permeabilized WT MIA PaCa-2 cells transfected with EV were set as one unit. Error bars indicated the mean ± SD of four independent experiments. **p < 0.01 versus EV, Student's t test. D, representative CRC traces of digitonin-permeabilized WT MIA PaCa-2 cells transfected with EV (black) or ATPIF1 (red) for 24 h in the presence of CsA. E, CRC/CRC 0 ratio of digitonin-permeabilized WT MIA PaCa-2 cells transfected with ATPIF1 normalized to those transfected with EV in the presence of CsA. CRC values of digitonin-permeabilized WT MIA PaCa-2 cells transfected with EV in the presence of CsA were set as one unit. Error bars indicated the mean ± SD of four independent experiments. ns, Student's t test. F, representative CRC traces of digitonin-permeabilized ΔPPIF MIA PaCa-2 cells transfected with EV (black) or ATPIF1 (red) for 24 h. G, CRC/CRC 0 ratio of digitonin-permeabilized ΔPPIF MIA PaCa-2 cells transfected with ATPIF1 normalized to those transfected with EV.CRC values of digitonin-permeabilized ΔPPIF MIA PaCa-2 cells transfected with EV were set as one unit. Error bars indicated the mean ± SD of four independent experiments. ns, Student's t test. CRC, calcium retention capacity; CsA, cyclosporine A; CyPD, cyclophilin D; EV, empty vector; IF1, mitochondrial ATP synthase inhibitory factor 1; ns, not significant; PTP, the mitochondrial permeability transition pore. [/fig]
[fig] Figure 5: IF1 interacts with p53-CyPD complex. A, HEK293T cells were transfected with EV or plasmids carrying ATPIF1 (encoding IF1 protein) or ATP5O (encoding subunit OSCP) for 24 h. Isolated mitochondria were lysed for co-IP and WB. The blots are representative of three independent experiments. [/fig]
[fig] Figure 7: p53 interacts with CyPD in an IF1-dependent manner. A, representative CRC traces of digitonin-permeabilized WT (black) and ΔATPIF1 (red) MIA PaCa-2 cells transfected with C terminus HA-tagged plasmids carrying indicated genes for 24 h. B, CRC/CRC 0 ratio of digitonin-permeabilized WT (black column) and ΔATPIF1 (red column) MIA PaCa-2 cells transfected with plasmids carrying the indicated genes normalized to those transfected with EV. CRC values of permeabilized WT (black) and ΔATPIF1 (red) MIA PaCa-2 cells transfected with EV were set as one unit, respectively. Error bars indicated the mean ± SD of five independent experiments. *p < 0.05, ns, two-way ANOVA with Bonferroni post hoc test. C, the effects of p53-CyPD complex overexpression on cell viability in WT and ΔATPIF1 MIA PaCa-2 cells revealed by ATP assay. Error bars indicated the mean ± SD of six independent experiments. **p < 0.01, ns, two-way ANOVA with Bonferroni post hoc test. D, WT and ΔATPIF1 MIA PaCa-2 cells were nontransfected or transfected with EV or with plasmids carrying ATPIF1 or PPIF or TP53 for 24 h. Protein was extracted for co-IP and WB. The blots are representative of three independent experiments. CRC, calcium retention capacity; CyPD, cyclophilin D; EV, empty vector; HA, hemagglutinin; IF1, mitochondrial ATP synthase inhibitory factor 1; IP, immunoprecipitation; ns, not significant; WB, Western blotting. [/fig]
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COVID-19 Prevalence in UNIVI Group Nursing Homes and Multilevel Geriatric Hospitals: Epidemiological Study of Immunological Status with Rapid Serological Tests for Diagnostic Guidance and Follow Up
BACKGROUND/OBJECTIVES: To date, data are lacking on the proportion of residents, and employees who have actually been exposed to SARS-Cov-2 in nursing homes and geriatric healthcare institutions, as well as the evolution of their serological status and the recurrence of Covid-19. The primary objective was to determine the prevalence of COVID-19 using NG Biotech rapid serological tests among caregivers and residents. The secondary objectives were to determine: prevalence according to RT PCR tests or clinical diagnosis; the risk factors (autonomy, arterial hypertension, diabetes mellitus) and clinical presentation (e.g. respiratory, abdominal or cutaneous symptoms, asthenia, fever) among residents; the risk factors (age, sex, profession, family situation) among caregivers; the evolution of the serological status at 1, 3 and 6 months using NG Biotech rapid serological tests; the symptomatic recurrence of Covid 19 at 1, 3 and 6 months. DESIGN: Multicentric prospective observational. SETTING: Study location: 27 nursing homes and 3 multilevel geriatric hospitals belonging to the UNIVI Group in France. PARTICIPANTS: 1334 professionals: 692 among multilevel geriatric hospitals (mean age: 43.6+/-11.8; 441 (82.4%) female) and 642 among nursing homes (mean age: 43.5+/-12.4; 685 (85.9%) female), and 1145 residents (mean age: 89+/-7.5; 898 (78.7%) female). MEASUREMENTS: Prevalence using NG Biotech rapid serological tests, medical diagnosis, RT-PCR tests.Risk factors among residents using the medical file and among caregivers using questionnaires. Clinical presentation in residents using the medical file. RESULTS: The prevalence using NG Biotech rapid serological test in residents was 14.4 % (168 of 1142 available diagnostics), the global prevalence (positive RT-PCR or positive serological test) was 22.7% (203 of 895 available diagnostics). The prevalence using NG Biotech rapid serological test in professionals was 12.8% (164 of 1315 available diagnostics), the global prevalence (positive RT-PCR test or positive serological test) was 23.8% (222 of 933 available diagnostics). The risk factors among residents were: living in an Alzheimer unit, and being a contact case. Being independent for activities of daily living was protective. The risk factor among caregivers was being a contact case. Another risk factor was the job; nurse assistants, nurses, and physicians were the most exposed. Residents had atypical clinical presentations including frequent geriatric syndromes (falls, delirium). 68.3% (71 of 104) of the initially positive residents still had a positive rapid serological test at 1 month follow up and 74 % (54 of 73) at 3 months follow up. 77.9% (88 of 113) of the initially positive employees still had a positive rapid serological test at 1 month follow up. Symptomatic reinfection was exceptional in caregivers or in residents during follow up.CONCLUSION: COVID 19 prevalence among caregivers and residents in nursing homes and geriatric health Institutions is underestimated when using only one method for diagnosis. Geriatric syndromes such as falls and delirium in residents should trigger further investigations on a COVID-19 cause. Immunity was persistent in ¾ of caregivers and residents during the 3 months follow up. The high prevalence of COVID 19 in geriatric institutions pleads in favor of the French vaccination policy, initially targeting as a priority the most vulnerable and dependent people, followed by staff members in healthcare institutions and nursing homes. More studies on the persistence of immunity and the perspective of Covid 19 mutations will help determine the long-term vaccine booster policy.
# Introduction
T he severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) was first reported in December 2019 in China. In February 2020, the Word Health Organization (WHO) named the disease caused by SARS-CoV-2 the 2019 Coronavirus disease (COVID-19) (1, 2).
In March 2020 the WHO declared the outbreak of a pandemic. COVID-19 rapidly spread in France, and was associated with high death rates, especially in older people [bib_ref] Coronavirus disease 2019 in elderly patients: Characteristics and prognostic factors based on..., Wang [/bib_ref].
In May 2020, The Epidemiology and Living conditions linked to COVID-19 study (EpiCov) was carried out in France. It included 370,000 randomly selected people. According to this study, 4.5% of the French population had a SARS-CoV-2positive serology. One of the risk factors of contamination was being a caregiver.
After the first COVID outbreak, the situation of the COVID 19 epidemic in geriatric healthcare institutions showed that both residents and caregivers had paid a heavy tribute and focused interventions were needed.
ELISA rapid serological tests can provide an assessment of immunological status (detection of IgG antibodies) in 15 minutes on a finger-prick blood sample. The French health authorities (Haute Autorité de Santé, HAS) (6) and the WHO (7) have validated use of these tests, notably for investigating on-going outbreaks, and for identifying previously infected individuals.
Residents and healthcare professionals of 30 geriatric healthcare institutions belonging to the UNIVI Group were invited to undergo ELISA rapid serological tests to ascertain their COVID-19 serological profile.
The primary objective of this study was to determine the prevalence of COVID-19 using NG Biotech rapid serological tests among caregivers and residents
The secondary objectives were to determine prevalence according to RT PCR tests or clinical diagnosis; the risk factors (autonomy, arterial hypertension, diabetes mellitus) and clinical presentation (e.g. respiratory, abdominal or cutaneous symptoms, asthenia, fever) among residents; the risk factors (age, sex, profession, family situation) among caregivers; the evolution of the serological status at 1, 3 and 6 months using NG Biotech rapid serological tests; the symptomatic recurrence of Covid 19 at 1, 3 and 6 months
# Methods
## Design
This was a prospective observational multicentric study conducted between July 24th 2020 and October 16th 2020 during a Covid-19 testing campaign launched by the UNIVI Group, a network of 71 healthcare institutions in France specialized in care for disabled and older persons. Participants in this study were recruited in the 27 nursing homes (1926 residents and 1165 employees), and the three multilevel geriatric hospitals (1092 employees) that constitute the UNIVI Group's geriatric institutions.
The Group decided to use an ELISA rapid serological test to ascertain the serological profile of the nursing home residents and caregivers and the multilevel geriatric hospital caregivers. NG Biotech finger-stick IgG-IgM combined antibody rapid tests were used [bib_ref] Analytical performances of five SARS-CoV-2 whole-blood finger-stick IgG-IgM combined antibody rapid tests, Péré [/bib_ref].
Residents and employees with a positive rapid serological test or a positive RT-PCR test were invited to attended followup visits at 1, 3 and 6 months for rapid serological tests.
## Participants
-All participants who had an ELISA rapid serological test were retained for analysis. -All residents of the 27 nursing homes, including those under legal guardianship, were invited to participate in the study. -All employees of the 30 healthcare institutions (27 nursing homes and 3 multilevel geriatric hospitals) were invited to participate in the study, irrespective of occupation. -As the objective of the study was to give an accurate as possible description of the population, no exclusion criteria were defined. -Results from vaccinated persons were not retained for analysis.
## Data collection
## Criteria for overall population
The results of three diagnostic strategies -clinical diagnosis, positive reverse transcriptase polymerase chain reaction (RT-PCR) results, serological tests -were collected. Positive diagnosis of infection since the beginning of the pandemic was recorded as follows: -probable SARS-CoV-2 infection based on symptoms and physical examination, -ongoing SARS-Cov-2 infection determined by a positive RT-PCR diagnostic test, -previous viral encounter determined by a positive NG Biotech rapid serological test.
The overall diagnosis of SARS-Cov-2 infection was considered positive if either the RT-PCR test or the rapid serological test was positive. Clinical assessment was not considered for determining the overall diagnosis. If the RT-PCR test data was unavailable and the rapid serological test negative, the overall diagnosis was not retained for analysis.
## Criteria specific to residents
Symptoms associated with a clinical diagnosis of probable SARS-CoV-2 infection were recorded. Potential risk factors were also noted: -Autonomy measured by Iso-Ressource group (GIR) which is a national scale to evaluate the degree of autonomy in older persons (GIR 1/2 : very dependent, GIR 3 / 4 : moderately dependent, GIR 5/6 : independent) -Arterial hypertension -Diabetes mellitus -Contact with an infected person All information was gathered from the resident's medical files.
## Criteria specific to staff members
Various potential risk factors were collected: -Age and gender -Profession -Type of housing (house or apartment) -Family context (single, couple without or with children, single-parent) -Mode of transportation (car, public transportation, walk/bike) -Contact with an infected person.
## Global data
The number of deaths among residents in each institution was collected from death certificate data for the period of March through May for the last three years (2018 to 2020).
## Follow-up data
Results of rapid serological tests performed at follow-up visits one, three and six months after inclusion were recorded for employees and residents who had an initially positive diagnosis of infection (RT-PCR test and/or rapid serological test).
Data on any new infections clinically suspected and confirmed by RT PCR test, were also collected.
## Ethics
The study protocol received approval before the beginning of the study from local authorities (Comité de Protection des Personnes Ile de France 1, N° IORG0009918). All participants (or the legal representative when appropriate) were informed about the study and those who declined to participate were not included.
The study was registered in clinicaltrials.gov (NCT04790994).
# Statistical analysis
Numeric data were described by mean and standard deviation (SD). Qualitative data were described by number and proportion of each modality.
The proportion of residents or employees with a positive test or diagnosis was computed.
To assess the impact of factors on the infection risk, mixed logistic models were used, including the factor as a fixed effect and the institution as a random effect. This was necessary because of considerable geographic differences in the pandemic situation.
A computing cross-table was used to search for a link between the clinical diagnosis and results of the RT-PCR or serological tests.
The number of deaths was compared between the years 2018, 2019 and 2020. A Poisson mixed model was used, with the period (COVID or not) added in fixed effect and the establishment in random effect.
At each visit, the proportion of positive rapid serologic test was computed, altogether with the number of new diagnostics of SARS-COV2 infections. The rates were compared according to the results of the initial test with Khi-2 tests or, in case of low numbers, with Fisher's exact tests.
A 5% significance threshold is considered. Analyses were carried out with R software (version 3.6.1).
# Results
## Population
In this study, 1145 residents (mean age: 89+/-7.5; 898 (78.7%) female) were included and 1334 professionals: 692 among multilevel geriatric hospitals (mean age: 43.6+/-11.8; 441 (82.4%) female) and 642 among nursing homes (mean age: 43.5+/-12.4; 685 (85.9%) female).
For residents, the sex-ratio (male to female) was 1:3.7 and the mean age was 89.0±7.5 years, ranging from 58 to 106 years. For staff members (considering all institutions), the sex-ratio was 1:5.5 and the mean age was 43.6±12.2 years, ranging from 18 to 73 years.
## Infection rate
The overall diagnosis was considered missing for 250 residents who had a negative rapid serological test and did not have an RT-PCR result due to unavailability [fig_ref] Table 1: Diagnosis of SARS-CoV-2 infection among residents and healthcare professionals using different methods [/fig_ref]. Among the 895 residents with an overall diagnosis, 203 (22.7 %) were confirmed positive for SARS-CoV-2 infection either by a positive RT-PCR test or a positive rapid serological test. Taken separately, the RT-PCR test, the rapid serological test, and clinical assessment each produced a lower infection rate than the overall diagnosis. The overall rate of SARS-Cov-2 infection in staff members was 23.8% (222/933); overall diagnosis data were missing for 401 [fig_ref] Table 1: Diagnosis of SARS-CoV-2 infection among residents and healthcare professionals using different methods [/fig_ref]. This rate was significantly higher (108/276; 39.1%) among multilevel geriatric hospital staff members than among nursing home staff members (114/657; 17.4%) (OR = 3.06 [2.20; 4.24]; p <0001). The rate of infection among staff members based on either RT-PCR test, the medical diagnosis or the serological test alone was lower than for the overall diagnosis.
Geographical differences were manifest: by institution, infection rates ranged from 2.9% to 71.1% for residents and 0% to 66.7% for caregivers.
## Risk factors
## Residents
Institutional residents were more likely to be confirmed positive for SARS-CoV-2 than staff members (odd ratio 1.67[1.24; 2.23] ; p = 0.001).
Prevalence among residents living in units designated as Alzheimer units was significantly higher than in conventional units (OR=1.85, p=0.048). The prevalence was significantly lower in residents who did not require assistance in activities of daily living (GIR 5-6) (p= 0.0024; GIR 1 or 2 vs GIR 5 or 6, OR= 0.22; GIR 3 or 4 vs GIR 5 or 6, OR= 0.21). Being a contact case was also a risk factor (p<0.0001) [fig_ref] Table 2: Covid-19 risk factors among nursing homes residents [/fig_ref].
## Staff members
For staff members, having been in contact with a COVID-19 case was a highly significant risk factor. The only other statistically significant factor was the occupational category. Exposure was greatest among nurse assistants, nurses and physicians [fig_ref] Table 3: Characteristic features of healthcare professionals by overall SARS-Cov-2 diagnosis as determined by... [/fig_ref].
## Clinical presentation in residents
Clinically, residents presented with asthenia and respiratory symptoms, 54 and 50% respectively. Fever was observed in only 31%. Geriatric symptoms were frequent: abdominal symptoms (24.5%), delirium (17.3%), and falls (12%) [fig_ref] Table 4: Characteristics of residents testing positive for SARS-Cov-2 [/fig_ref].
## Lethality
The number of deaths for the period March to May was significantly higher in 2020 than in 2018 and 2019. The number of deaths increased 1.52-fold (p<0.0001), with 150 deaths in 2018 (data were not available for three institutions), 135 in 2019 and 230 in 2020.
## Follow-up visits
Follow-up visits were scheduled for 203 residents, of whom 137 (67.2%) attended the 1-month visit, 90 (44.1%) the 3-month visit and 12 (5.9%) the 6-month visit.
Follow-up visits were scheduled for 222 employees, of whom 147 (66.2%) attended the 1-month visit, 99 (44.6%) the 3-month visit and 28 (12.6%) the 6-month visit.
Due to the concomitant vaccination campaign that had started before the scheduled 6-month visit, few employees attended; thus, the 6-month results are not reported.
## Time course of rapid serologic tests
There was a strong link between the initial rapid serologic test result and the presence of antibodies at each visit. Among residents with an initial positive test, 68% still had a positive test at 1 month, and 74% at 3 months. For employees with an initial positive test, 78% still have a positive test at 1 month and 54% at 3 months [fig_ref] Table 5: Results of serological tests among residents and employees during follow-up [/fig_ref].
## Reinfection
At 1 month, the rate of clinically symptomatic reinfection was low: 1.5% suspected infection in residents (n=2) and 2.1% in employees (n=3); rate of RT-PCR confirmed infection = 2.1% in employees (n=3). At 3 months, the rate of suspected infection in residents was 1% (n=1) and 3.3% in employees (n=3); rate of RT-PCR confirmed infection = 3.1% in employees.
# Discussion
The overall prevalence of COVID-19 infection among residents between the two waves of the pandemic in France (May to August 2020) was 22.7% while the seroprevalence was 14% and the RT-PCR prevalence 13%.
In a study determining COVID-19 prevalence by RT-PCR in 24 nursing homes in the Marseille area of France, the prevalence was 13.4% (10), a level similar to the 12.9%
RT-PCR prevalence in our study. In our opinion, these data underestimate the global impact of COVID-19 that is better described taking into account both RT-PCR results and serology. We observed that clinical diagnosis of COVID-19 was poorly adapted to an estimation of overall prevalence, mainly because older people present with comorbidities and frequent cognitive impairment, which makes COVID-19 diagnosis quite challenging.
Our different estimations of COVID-19 prevalence among institutional residents are all higher than the 5.7% overall positivity rate among dependant residents of French institutions reported by a national survey (39,638 cases in nursing homes on July 31st 2020 among an estimated 695,060 elderly dependent residents of French nursing care institutions). This may be because most of the UNIVI Group nursing homes are situated in high COVID-19-prevalence zones in France.
Regarding caregivers, the EpiCov study among people working in French healthcare institutions in May 2020 found that 4% were positive for SARS-Cov-2 (ELISA method). The prevalence in our staff members was higher (23.8 %). This may have been because the EpiCov study took into consideration staff less frequently in contact with patients positive for SARS-Cov-2 (laboratory technicians, ambulance personnel, etc). This may also be because we pooled RT-PCR test and rapid serological test results. The risk factor of a large number of people living together noted in the EpiCov as a risk factor of COVID-19 was not observed in this study. Statistical power may be an issue here.
Regarding prevalence in both staff members and residents, the NG Biotech rapid serological tests appeared to have sufficient sensitivity for identifying individuals with past SARS-CoV-2 infection. Nevertheless, some rapid serological tests may lack specificity, with a risk of false positivity mainly on the IgM band, which may lead to an overestimation of the seroprevalence [bib_ref] Analytical performances of five SARS-CoV-2 whole-blood finger-stick IgG-IgM combined antibody rapid tests, Péré [/bib_ref]. Regarding the risk factors for infection, the prevalence in residents living in units designated as Alzheimer units was significantly higher. Moreover, COVID-19 prevalence was significantly lower in residents who did not require assistance in activities of daily living (GIR 5-6). This suggests a higher risk of COVID-19 infection in residents with severe cognitive impairment with behavioral disturbances, probably due to poorer compliance with barrier gestures and greater frequency of medical staff assistance. It should also be noted that the COVID-19 symptomatology presented by the elderly residents was dominated by asthenia (53.6%) and respiratory symptoms (49.5%) with a nonnegligible frequency of digestive symptoms (diarrhea, etc.). Geriatric syndromes (delirium, falls) have been described as a possible clinical presentation in older patients. Geriatric syndromes and fever were found in one-third of cases. Fever was less frequent than in other studies describing clinical characteristics of older patients .
Follow-up was in a favor of a satisfactory rate of persistent immunity in three-quarters of the study population, both caregivers at one month and residents at 1 and 3 months according to the rapid serological test that does not take into account T cell-linked immunity.
Concerning reinfection, Firzan et al. [bib_ref] SARS-CoV-2 reinfection and implications for vaccine development, Nainu [/bib_ref] described a number of cases at an apparently higher rate than in our study, perhaps related to the fact that reinfection was only suspected in symptomatic caregivers and residents.
Our study has some limitations. The population was not randomly and homogeneously recruited. The use of individual preventive measures by caregivers outside of work was not documented which may have caused bias. The residents tested had survived COVID-19 first wave and were therefore more robust than the usual nursing home population.
There also is a significant number of residents and staff members with missing RT-PCR results, eventually related to the fact that RT-PCR was scarcely available at the beginning of the COVID-19 pandemic. When a missing RT-PCR result was associated with a negative rapid serology result, the overall infection diagnosis was considered missing.
Missing data during follow-up was also noted, but probably occurred because vaccination policy was a priority, inducing many vaccinated residents to drop out of the study. For employees, who adhered less well to follow-up, the results are only statistically significant at 1 month.
Moreover, even though the UNIVI Group has a large number of nursing homes, they are not located in all regions of France, which limits geographical generalization.
Furthermore, limitations related to informing the most cognitively impaired may have created a selection bias. Nevertheless, we were able to include nursing home residents with different levels of autonomy.
Another limitation concerns the difficulty of diagnosing COVID-19 in older residents. In order to limit this bias, we chose to combine the RT-PCR results with the rapid serological test results in order to increase diagnostic sensitivity.
Finally, mortality rate could not be determined. We can nevertheless hypothesize that the higher mortality observed in 2020 was due to the COVID-19 pandemic.
# Conclusion
This is the first epidemiological study carried out in residents and caregivers of 27 nursing homes and 3 French multilevel geriatric hospitals between the 2 epidemic waves of the SARS-COV-2 pandemic. The results provide an estimate of COVID-19 prevalence based on different diagnosis methods. The seroprevalence determined by rapid serological tests in institutional residents was 14%, an underestimation compared with the overall prevalence of 22.7%. In staff members, the seroprevalence determined by rapid serological tests was 12.8%, again underestimated compared with the overall 23.8% prevalence.
Geriatric syndromes such as falls and delirium were frequent and should be taken into account as a possible clinical presentation of COVID-19 in order to trigger further investigations. Clinicians should also be aware of such manifestations of COVID-19 in order to institute preventive measures.
For institutional residents, living in an Alzheimer unit was found to be a risk factor for COVID-19. Inversely, independence for activities of daily living was a protective factor. For caregivers, having been in contact with a COVID-19 infected person was the major risk factor.
Immunity seemed to be persistent in three-quarters of caregivers and residents during the 3-month follow-up.
The high prevalence of COVID-19 in geriatric institutions pleads in favor of the French vaccination policy, initially targeting as a priority the most vulnerable and dependent people, followed by staff members in healthcare institutions and nursing homes. More studies on the persistence of immunity and the perspective of COVID-19 mutations will help determine a long-term vaccine booster policy.
## Conflict of interest:
The authors have no conflict of interest.
Author Contributions: A. de Malherbe participated in the study concept and design acquisition of subjects and data, interpretation of data and preparation of the manuscript; S. Verdun participated in the study concept and design, analysis and interpretation of data, and preparation of the manuscript; V. Brenière participated in the acquisition of subjects and data, interpretation of data and preparation of the manuscript; M. Jourdan participated in the acquisition of subjects and data and preparation of the manuscript; L. Luquel participated in the acquisition of subjects and data and preparation of the manuscript; M. Harboun participated in the study concept and design acquisition of subjects and data, interpretation of data and preparation of the manuscript.
Sponsor's Role: UNIVI financed the exploitation of the eCRF, the recruitment of two nurses who contributed in collecting data in nursing homes. UNIVI also financed the rapid serological tests.
[table] Table 1: Diagnosis of SARS-CoV-2 infection among residents and healthcare professionals using different methods: RT-PCR test, serological test and medical diagnosis [/table]
[table] Table 2: Covid-19 risk factors among nursing homes residents. Overall diagnosis determined by positive RT-PCR and/or serological test [/table]
[table] Table 3: Characteristic features of healthcare professionals by overall SARS-Cov-2 diagnosis as determined by RT-PCR and/or rapid serological tests [/table]
[table] Table 4: Characteristics of residents testing positive for SARS-Cov-2 [/table]
[table] Table 5: Results of serological tests among residents and employees during follow-up [/table]
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Selection and analytical applications of aptamers binding microbial pathogens
DNA aptamers specifically recognizing microbial cells and viruses have a range of analytical and therapeutic applications. This article describes recent advances in the development of aptamers targeting specific pathogens (e.g., live bacteria, whole viral particles, and virally-infected mammalian cells). Specific aptamers against pathogens have been used as affinity reagents to develop sandwich assays, to label and to image cells, to bind with cells for flow-cytometry analysis, and to act as probes for development of whole-cell biosensors. Future applications of aptamers to pathogens will benefit from recent advances in improved selection and new aptamers containing modified nucleotides, particularly slow off-rate modified aptamers (SOMAmers).
# Introduction
Infectious diseases are the leading cause of death worldwide, and rank as the third highest cause of death in Canada and the USA after malignant neoplasms and cardiovascular disease. The rapidly growing global population, the rise of antimicrobial resistance, and recent resurgence in the incidence of infectious disease, most notably tuberculosis and smallpox, highlight the severity of the threat.
Pathogen surveillance is critical to diagnosis, control, and prevention of infectious diseases. Technology in the area of routine pathogen detection in the laboratory and the clinic has been slow to develop. Common diagnostic and surveillance methods include culture, biochemical tests, and antibodybased methods (e.g., agglutination and enzyme-linked assays). Definitive identification involves the amplification-based methods of regular and real-time PCR (quantitative PCR, or qPCR). Although fast and sensitive, PCR and qPCR are too complex to carry out in a basic clinical laboratory setting lacking highly-trained personnel. There is continued need for point-of-care assays that are simple, sensitive, and economical.
Aptamers are a class of flexible reagents with the potential to play a role in pointof-care assays. This group of synthetic nucleic-acid molecules can be generated in vitro against virtually any target molecule. The affinity of aptamers for their targets is comparable to, or even higher than, most monoclonal antibodies. Typical dissociation constants for aptamer-target complexes are found in the picomolar to low micromolar range. Aptamers possess key advantages over antibodies due to their increased flexibility and the nature of in vitro evolution. They have low variability between batches, do not rely on animals for their production, can be created against toxic or non-immunogenic targets, are economical and fast to produce, and can be synthesized against a complex target without prior knowledge of a specific target molecule or without purifying a known target molecule.
Aptamers can be chemically modified and incorporated into a variety of simple assays for pathogen detection, as well as more complex assay formats, including flow cytometry, cell imaging, and aptamerbased biosensors. Aptamers have long shelf-lives and they can be used to coat surfaces and devices for point-of-care testing applications. These devices could potentially be re-useable after heating due to the thermal stability of aptamers.
This article discusses aptamer technology pertinent to pathogenic targets. The objective is two-fold:to summarize and to evaluate recent developments in the selection of aptamers against pathogens; and,to evaluate the analytical applications of these aptamers.
## Selection of aptamers against pathogenic targets
2.1. Selection of aptamers against purified molecular targets Aptamers can be evolved via an iterative process called Systematic Evolution of Ligands by Exponential Enrichment (SELEX). Different SELEX methodologies have been previously reviewed. Briefly, a singlestranded random DNA or RNA library containing 10 14 -10 16 unique sequences is incubated with a target of interest. The target-bound and unbound nucleic-acid sequences are separated, and the target-bound sequences are amplified for use as inputs in the next round of selection. The random nucleic-acid library typically contains 40-100 nucleotide (nt) single-stranded sequences with a randomized stretch of nts in the center and fixed primer sequences on either end. As many as 20 rounds of selection are carried out until a pool of aptamer sequences with high target affinity is obtained. These aptamers can then be cloned and sequenced. Most aptamers selected against pathogenic targets have been evolved using purified target molecules, using the common SELEX procedures [fig_ref] Figure 1: Conventional SELEX for generating DNA aptamers [/fig_ref]. It is easier to obtain high-affinity aptamers in fewer rounds of SELEX with a purified target. Various bacterial or viral molecules have been used, mostly proteins. Carbohydrates have also been targeted including the lipopolysaccharide (LPS) of E. coli O111:H4 and cell-surface extracts of C. jejuni. [fig_ref] Table 1: Purified pathogen molecules that have been used in selection of aptamers by... [/fig_ref] summarizes molecules purified from pathogens against which aptamers have been selected. The use of amino-acid side-chain mimics in nt structure is a recent breakthrough that has allowed aptamers to be selected against previously impossible protein targets. In the future, aptamers incorporating such chemical modifications will probably expand the SELEX target repertoire even further.
## Selection of aptamers against live cells
Although aptamers can be selected using purified target molecules, their applications to target whole live cells may sometimes be limited, because the binding sites of cell-surface molecules can be different from their isolated forms. As a result, whole live cells have become increasingly common SELEX targets. SELEX using whole cells allows molecules to be targeted in their native conformations on the cell surface. It also negates the need for complex purification and target-partitioning. A random DNA library, typically containing 10 14 -10 16 unique sequences of single-stranded oligonucleotides, is incubated with the target molecule of interest. The target-bound and unbound sequences are separated, and the target-bound sequences are amplified by PCR for use as inputs in the next round of selection. The reiterated rounds of selection are carried out to generate a pool of aptamer sequences that have high affinity for the target molecule. These aptamers are then cloned and sequenced.
## Trends
Trends in Analytical Chemistry, Vol. steps. Cell-SELEX against mammalian cells has been extensively reviewed. Bacterial cells present unique challenges over mammalian cells due to their cell-wall structure. Gramnegative bacteria possess a negatively-charged outer membrane that can repel nucleic-acid molecules. Capsules covering the surface of many pathogenic bacteria can also obscure target proteins. Both of these factors make it difficult to generate aptamers that bind to the surface molecules of bacterial cells. In addition, bacteria often grow much faster than culture cells of mammalian tissue. Short generation times lead to rapid changes in protein expression and high surface variation between cultures and colonies. Such variation can impede consistent measurement of aptamer binding.
The bacterial cell-SELEX procedure developed by our group is described in [fig_ref] Figure 2: Bacterial cell SELEX[75] [/fig_ref]. Briefly, pathogenic bacterial cells are incubated in solution with a randomized nucleic-acid library. Cell-bound nucleic acids are separated from unbound nucleic acids via centrifugation. Cells are then washed, and cell-bound nucleic acids are eluted using low salt concentrations at high temperature. Cell-bound aptamers can also be amplified directly from the cell surface. Aptamer pools obtained after each round are screened for increased target-cellbinding affinity (e.g., by flow-cytometry analysis). Rounds of counter-selection against non-target cells, in which the sequences bound to the nontarget cells are removed, can be introduced after the first two rounds of positive selection. Alternatively, positive selection and counter-selection rounds can be alternated. These studies demonstrated the feasibility of selecting aptamers for both Gram-positive and Gram-negative bacteria. A summary of pathogenic whole cell and whole virus targets is presented in [fig_ref] Table 2: Whole bacterial cells and viral particles used in selection of aptamers by... [/fig_ref].
As another important feature of whole cell-SELEX, specific target molecules do not need to be identified or purified ahead of time. Aptamers binding to the cell surface can be used to purify and to identify their respective target molecules post-SELEX. For example, aptamers against cell-surface molecules conjugated to magnetic beads can be used to pull the target out of a cell-lysate or crude-membrane preparation via either affinity interaction or photocrosslinking. Once the aptamer-protein complexes are eluted from the magnetic beads, they can be electrophoresed and identified via mass spectrometry.
While the majori of work in post-SELEX target elucidation has been done with mammalian cells, the principles also apply to whole-cell pathogens. Aptamers derived against the surfaces of whole African Trypanosomeswere used to identify continuously expressed proteins in the variant surface coat of Trypanosoma brucei including a 42-kDa protein in the flagellar attachment zoneand a variant surface glycoprotein. Our group has identified a putative target of a Lactobacillus acidophilus specific DNA aptamer as the S-layer protein. We have also identified putative target molecules of several aptamers selected against Group A Streptococcus [80].
## Analytical applications of selected aptamers
Aptamers have proved useful in a variety of assays that conventionally use antibodies, including ELISA-like assays, western blotting, flow cytometry, microscopy, affinity chromatography, and capillary electrophoresis. Also, many novel technologies (e.g., aptamer-based biosensors and nano-devices, and ligation and amplification assays) are developed by taking advantage of aptamers. Today the applications of aptamers derived via whole cell-SELEX are limited, mainly because the number of aptamers derived against viral or bacterial targets is limited. Following is a summary of whole-cell aptamer technologies that have potential applications against pathogens, or have already been applied for pathogen detection.
## Sandwich assays
In a traditional sandwich assay, an antibody is immobilized on a solid surface. Upon target binding, a secondary antibody attached to an enzyme (ELISA) or fluorophore is added. The secondary antibody binds to a different epitope on the target molecule than the primary antibody. Several sandwich assays using aptamers in place of antibodies have been developed to measure bacterial antigens.
An aptamer-linked immunosorbent assay was developed for detection of Francisella tularensis antigen. A mixture of 25 different aptamers was used as both a capture and detection reagent. An aptamer cocktailcoated micro-titer plate was incubated with bacterial antigen, after which a biotin-labeled aptamer cocktail was added and the bound antigen detected via streptavidin-horseradish peroxidase. The aptamer assay demonstrated specificity and affinity comparable to that of an antibody-based ELISA.
The sandwich format can also be used to screen an aptamer pool for candidates with high affinity and selectivity to pathogenic antigens. A selected ssDNA aptamer population specific for Leishmania infantum histone H2A protein was screened using purified recombinant H2A-coated microtiter-plate wells. Recovered aptamers were found to be specific for H2A after only three SELEX rounds.
## Flow cytometry
Flow cytometry is a common method for cell sorting, counting, and detection. Cells are passed through a flow cell individually, and forward and side-scattered light intensities are measured. A laser beam is also used to excite fluorescent molecules, and fluorescence intensity is measured. Hence, cells bound to a fluorescent probe or dye can be identified and sorted from the population.
Antibodies are often used as probes, but aptamers have been found to be equally efficient. In addition to target identification and purification, aptamers are useful probes for identification and collection of cells expressing a target molecule of interest. Flow cytometry has been used both to evaluate target recognition by an aptamer pool and to isolate cell populations expressing a target. However, there are difficulties in using aptamers for cell typing in this fashion, as discovered in a standardized flow-cytometry assay by the Ellington group. This problem is also faced when using antibodies in cell typing. There is not always a correlation between binding to the cell and biomarker expression on the cell surface; sometimes, the cell-SELEX procedure selects for sequences with high affinity but poor specificity.
Specific aptamer sequences can be fluorescentlylabeled and used in tandem to sort bacterial cells or virus-infected cells. Cells are identified based on a distinct pattern of multiple aptamer binding. Closely-related cells give their own distinct patterns of binding to aptamers, and aptamers evolved against different cells can identify specific cells in a population. For example, SELEX carried out on vaccinia-infected cells generated a panel of aptamers with high affinity to cells expressing viral proteins. When used together in flow-cytometric analysis, these aptamers could identify vaccinia infection in a variety of cell lines. The aptamers also generated distinct intensities and patterns of binding dependent on the strain of vaccinia.
Another study selected aptamers against whole Staphylococcus aureus cells and used a fluorescently-labeled panel of five high-affinity aptamers to differentiate different S. aureus strains. The aptamers were shown to bind different cell-surface targets via a competitive flow-cytometry experiment; using five aptamers combined, rather than individually, was superior at detecting bacteria in pyogenic fluid.
Another useful way to employ flow cytometry is in determination of binding affinity between the target and the aptamer pool obtained from each round of selection. Bacterial cell-SELEX targets against which this technique has been applied include L. acidophilus, M. tuberculosis, C. jejuni, S. aureus, P. aeruginosa, and S. pyogenes. Recently, flow cytometry was used to screen aptamers that are specific for vaccinia-virus infected A549 cells.
Conjugation of aptamers to fluorescent nanoparticles or nanorods allows for increased fluorescence intensity over regular fluorescent labels when cells are analyzed via flow cytometry, and hence increased sensitivity of detection. Using fluorescent nanorods as aptamer scaffolds for flow cytometry can potentially render useful medium-affinity aptamers derived from cell-SELEX as assembly of aptamers on the nanorod surface increases their binding affinity.
## Aptamer biosensors
All biosensors comprise a biological molecularrecognition element (MRE] and a signal-transduction element (STE). A classical biosensor functions to transduce target binding by the MRE into a readable output. The term aptasensor applies to biosensors in which an aptamer is used as one or both of these elements. Aptasensors have seen increasing use for detection of pathogens in environmental and clinical samples. An excellent review of some of these methods has been published.
A variety of technologies have been applied to the development of aptasensors for pathogen detection. The earliest aptasensors for bacterial detection were based on an electrochemi-luminescence (ECL) sandwich format. SELEX was carried out using heat-killed anthrax spores as a separation matrix. Resultant ssDNA aptamers with high affinity for the spores were conjugated to magnetic beads or to biotin. The magnetic beadaptamer conjugates acted as capture reagents and the biotinylated aptamers as reporters for detection of as few as 10 spores with a linear dynamic range of 10-6x10.
Aptasensors with fluorescent labels were also used for pathogen detection. The same group that created the ECL anthrax sensor designed another sensor by conjugating aptamers selected against Bacillus thurigensis spores to CdSe quantum dots. This assay could detect as few as 1000 CFU/mL of spores.
Quantum dots were also integrated into a sandwich assay with aptamer-conjugated magnetic beads for detection of Campylobacter jejuni. Surface proteins extracted from C. jejuni were used as SELEX targets and the two highest affinity resultant aptamers were used as capture and detection ligands. The capture aptamermagnetic bead conjugates could be adhered to the surface of a cuvette via a magnet, for uniform, reproducible laser excitation. Unlike the previous sensor for anthrax, this sensor detected both live and heat-killed cells at levels as low as 2.5 CFU in complex food matrices. The assay was also portable; fluorescence could be measured via bench-top or hand-held fluorometers.
A FRET-based assay was developed for detection of a peptide from foot and mouth disease (FMD) virus VP1 structural protein. A polyclonal mixture of aptamers was labeled with Alexa Fluor 546-14-dUTP and incubated with quencher-bound FMD peptide. Binding of the FMD peptide to the aptamers resulted in a ''lightsoff'' response that could measure levels of FMD peptide as low as 25 ng/mL. The peptide-bound aptamers were also fixed to a solid support, and a competitive assay with introduced analyte resulted in a ''lights-on'' response.
A similar approach was used to detect Escherichia coli using aptamers evolved against E. coli outer membrane proteins (OMPs). Aptamers were labeled with Alexa Fluor, and E. coli cells were heavily labeled with Black Hole Quencher (BHQ). Fluorescent aptamer/BHQ-labeled bacteria complexes were attached to a microtiter plate; upon introduction of unlabeled E. coli cells, the quencher-labeled cells were competed away and a measurable signal was generated. This method could detect as few as 30 unlabeled live cells.
A DNA capture-element (DCE) sensing system based on fluorescence quenching was also developed by this group for both aptamer selection and sensing of B. thurigensis, B. anthracis spores, botulinum neurotoxin and MS-2 bacteriophage. Magnetic beads were attached to a dual fluorescently-labeled and quencher-labeled DNA aptamer; upon interaction of the aptamer with a target cell, the dsDNA is denatured into ssDNA. This dissociates the quencher and generates a fluorescent signal. The magnetic bead-aptamer-target-cell complexes can then be purified out of solution and redetected with a complementary DNA probe attached to a quencher.
Recently, a fiber-optic sandwich biosensor was created using a fluorescently-labeled detection aptamer and an antibody-coated optical waveguide surface for detection of Listeria monocytogenes in food. Fiber-optic biosensors measure the interaction between cells and aptamers by detecting waves emitted via excitation of a fluorescent reporter, usually an antibody. However, aptamers are ideal reagents for inclusion in such sensors. Due to their small size, aptamers exhibit high binding density on the cell surface, yielding enhanced signal and
## Trends
Trends in Analytical Chemistry, Vol. sensitivity. The DNA aptamer was fluorescentlylabeled and found to be specific for Internalin A, an invasion protein and pathogenicity marker. This sensor could detect as few as 10 3 CFU/mL of L. monocytogenes, and was successful at detecting cells spiked into chicken, beef, and turkey. Several novel aptasensors relying on fluorescently-labeled aptamers have been developed for unlabeled Hepatitis C virus (HCV). One is a chip-based diagnostic sensor for HCV made from sol-gel immobilized anti-HCV core protein aptamers on a 96-well plate. A secondary fluorescently-labeled anti-core antibody was used for detection. HCV particles in infected patient sera could be detected; normal patient samples did not generate a signal.
Aptamers do not need to be fluorescently-labeled for use in pathogen sensing. Their presence can be also detected via PCR amplification. Aptamers were selected against Salmonella typhimurium OMPs and conjugated to magnetic beads for collection of S. typhimurium from chicken-carcass rinse samples and subsequent quantitative PCR detection. The assay had a limit of detection (LOD) of 10-100 CFU/mL of rinsate. A recirculation format of the assay was created by holding the aptamer-coated magnetic beads in place on a tube with a capture magnet, and running diluted chicken rinse through. The LOD of the recirculation assay was 4-40 CFU/mL of rinsate; both assays allow for sample processing and work on complex chicken-litter samples.
Similarly, antibody-coated magnetic beads were used to collect E. coli from complex samples with detection via PCR amplification of an E. coli-specific aptamer. The assay could detect as few as 10 cells/mL of sample with a linear dynamic range of the order of 10-10 7 E. coli/mL, and was specific for the target cells.
Electrochemical detection has recently been applied to bacterial detection, specifically in the design of a probe for Salmonella Typhi detection in complex food samples. This probe method is rapid, portable, and label-free. The electrodes are also reusable after reconstitution in buffer. A single-walled carbon nanotube (SWCNT) layer is deposited on a glass-rod electrode. The layer is coated with an aptamer specific for S. Typhi type IV pili. Conformational changes induced by target cell binding to the aptamers result in a change of charge on the SWCNTs and a subsequent change in potential recorded by the electrode. This system could detect below 1 CFU/ mL of sample in less than 1 min with a linear dynamic range of 0.2-10 3 CFU/mL.
Another group developed a resonance sensor that could detect HCV helicase at levels as low as 100 pg/mL. The principal components of this sensor are nanomechanical microcantilevers. RNA aptamers specific to HCV helicase are attached to the cantilever sur-face; aptamer-target binding induces stress on the cantilever surface and a dynamic response change that can be measured.
## Cell labeling and imaging
Aptamers and aptamer-nanoparticle conjugates can be labeled and used in microscopy for detection of target cells. Fluorescence microscopy is recommended as a confirmatory method to be used in addition to flow cytometry or fluorescence measurement for aptamerbased cell typing. Microscopy can be carried out with fluorescently-labeled aptamers and aptamernanoparticle conjugates.
Aptamers specific for Trypanosoma cruzi were used as probes to label the parasite surface fluorescently. Fluorescence in-situ hybridization (FISH) with fluorescently-labeled DNA aptamer probes has been successful at imaging Pseudomonas aeruginosa. Optical microscopy has also been used to visualize the density of cells captured by aptamers on a microfluidic device.
However, there are instances where the target cell/ aptamer interaction cannot be microscopically visualized. For example, a modified avidin method could detect spores binding to aptamer-coated magnetic beads; this interaction could not be visualized under the microscope-most probably due to the hindrance from the spacing of the aptamers on the beads and the size of both the beads and the spores.
While it is easier for aptamers to label molecules on the cell surface, they have been used for intracellular imaging. Imaging of bacterial RNA in live cells (E. coli) has been accomplished using protein complementation regulated by the interaction of an RNA-binding protein and an aptamer. The RNA-binding protein is engineered to express GFP only in the aptamer-bound state; mRNA and rRNA can be tagged with aptamer and localized.
## Therapeutic potential of aptamers
The inhibitory activity of certain aptamers on their target molecules has therapeutic ramifications, particularly for cell-surface molecules and receptors. Most research on therapeutic aptamers has focused on cancer therapy.
A number of inhibitory aptamers have been isolated for pathogens, including Trypanosoma cruzi, Hepatitis C Virus, SARS coronavirus, E. coli lipopolysaccharide (LPS), and M. tuberculosis polyphosphate kinase 2.
Also, many of the aptamer-based technologies developed for chemotherapeutic drug delivery to a host cell surface could be modified for use with antimicrobials.
## Somamer arrays
The advent of slow off-rate modified aptamer (SOMAmer) technologyhas the potential to extend the applicability of aptamer-based pathogen surveillance, detection and sensing formats. Gold and colleaguesdeveloped a new generation of aptamers, termed SOMAmers, with two key innovations, utilizing chemically modified nts to generate SOMAmers with protein-like properties and employing kinetic manipulations to improve the specificity of SOMAmers. shows a SOMAmer-selection process. The process begins with incorporation of modified nts (four dUTP analogs) into the DNA library by polymerase extension of a primer annealed to a biotinylated template. After separation from biotinylated templates, the DNA library containing modified nts is incubated with the target molecule. The slow off-rate enrichment and partition process is subsequently performed with the partition step of high stringency, including the use of extensive washing with large volumes. After separation of the bound DNA from the unbound DNA, the enriched DNA sequences are amplified by PCR to prepare the DNA pool for the next round of selection. Because the chemically-modified dUTP analogs in the modified DNA library and the enriched DNA pools contain functional groups that mimic amino-acid side-chains, the SOMAmers generated are thought to have some ''protein-like'' properties. The expanded chemical diversity due to the incorporation of the modified nts extends SELEX to selection of aptamers against more difficult targets (e.g., carbohydrates on microbial cell surface and wide range of protein targets). Indeed, huge potential has been demonstrated from the success of obtaining aptamers (with pool Kd values below 30 nM) against 1200 human proteins.
SOMAmers were applied to develop a new proteomic array technology by taking advantage of both proteinbinding and complementary base-pairing features of aptamers. Biotin-conjugated SOMAmers were first incubated with samples and SOMAmer-protein complexes were then captured onto streptavidin-coated beads. After washing the beads, target proteins in complexes were then modified with a NHS-biotin tag. The complexes were photocleaved from the first set of beads and challenged with a polyanionic competitor (dextran sulfate) for selective disruption of non-specific binding interactions. The complexes were further attached via the target protein biotin to a second set of avidin-coated beads. After the free SOMAmers were washed away, the bound SOMAmers were released from their targets and quantified. The number of SOMAmers was directly pro- . SOMAmer selection technique. Four dUTP analogs were modified with chemical groups that mimic amino-acid side-chains. These modified nucleotides (nts) were incorporated into the DNA library by polymerase extension of a primer annealed to a biotinylated template. After separation from biotinylated templates, the single-stranded DNA library containing the modified nts was incubated with the target molecule. The slow off-rate enrichment and partition process involving high stringency was used to separate and to enrich the DNA sequences that were bound to the target molecule. The bound DNA sequences were amplified by PCR and were used as new DNA pool for the next round of selection. The reiterated rounds of selection were carried out to generate a pool of SOMAmers that have high affinity for the target molecule.
## Trends
Trends in Analytical Chemistry, Vol. portional to the number of target molecules (i.e. one SOMAmer per analyte). The utility of the SOMAmer assay was demonstrated by measuring 813 different proteins with pM levels of LODs and 7 logs of overall dynamic range. SOMAmer arrays have been successfully applied to identify novel biomarkers and proteome profiles in chronic kidney disease and non-small-cell lung cancer. Both SOMAmer selection and array technology could potentially be developed for pathogen surveillance, typing of pathogen species and strain, and detection of microbial infection. They could enable comparison of proteome profiles between pathogens or infected and healthy individuals. Another exciting application could be high-throughput, multiplex antimicrobial resistance screening or comparison of antibiotic resistance profiles of different bacterial species or strains. Also, comparison of proteome profiles between patients could be used for diagnosis of infection and identification of potential treatment options.
## Concluding remarks
Aptamers are an emerging class of reagents exciting due to their flexibility. They can be integrated into a myriad of existing and novel assay formats, and can be selected to have affinity and selectivity for virtually any target. The recent development of SOMAmer technology has opened up new targets to SELEX.
Although initially slow to develop, selection of aptamers against bacterial and viral targets has grown steadily in recent years. Such targets include purified cell-surface components and whole cells. Aptamers have shown promise in pathogen detection, collection, and therapy. Existing diagnostic and detection technologies have been modified to include aptamers targeting bacteria and viruses. In addition, a variety of novel aptamer-based assays have been developed for pathogen detection. Many existing aptamer-based assays initially designed for cancer cells could also be used for bacterially or virally-infected cells. The assays simply need to be modified to include aptamers against pathogenic targets in lieu of cancer-related targets.
SOMAmer technology has also demonstrated the feasibility of selecting aptamers against more than 1000 targets and of aptamer arrays that can simultaneously detect hundreds of targets. Such aptamer-based assays for pathogenic detection can fill the existing need for rapid, reliable point-of-care testing in both the laboratory and the clinic. Also, aptamers can be extended towards much needed antimicrobial and antiviral applications.
[fig] Figure 1: Conventional SELEX for generating DNA aptamers [/fig]
[fig] Figure 2: Bacterial cell SELEX[75]. A random DNA library, typically containing 10 14 -10 16 unique sequences of single-stranded oligonucleotides, is incubated with the target bacterial cells. The mixture is centrifuged and the cells are washed to remove the unbound sequences. The cell-bound DNA sequences are eluted off, amplified by PCR, and used as a new DNA pool for the next round of selection. The reiterated rounds of selection are carried out to generate a pool of aptamer sequences that have high affinity for the target cells. These aptamers are then cloned and sequenced. [/fig]
[table] Table 1: Purified pathogen molecules that have been used in selection of aptamers by SELEX [/table]
[table] Table 2: Whole bacterial cells and viral particles used in selection of aptamers by SELEX [/table]
|
Surgical Management of Hepatoblastoma and Recent Advances
Hepatoblastoma is the most common childhood liver malignancy. The management of hepatoblastoma requires multidisciplinary efforts. The five-year overall survival is approximately 80% in developed countries. Surgery remains the mainstay of treatment for hepatoblastoma, and meticulous techniques must be employed to ensure safe and effective local control surgeries. Additionally, there have been several advances from both pediatric and adult literature in the way liver tumor surgery is performed. In this review, we highlight important aspects of liver surgery for hepatoblastoma, the management of metastatic disease, and the most current technical advances in performing these procedures in a safe and effective manner. 85% of the patients with primary liver transplantation[10]. These achievements in hepatoblastoma treatment are the results of joint international efforts which have led to the development of treatment guidelines. Transplantation for hepatoblastoma has been recently reviewed[11]. In this review, we mainly focus on the surgical resection of hepatoblastoma.Preoperative PlanningA variety of radiographic tools can be used to image hepatoblastoma, and surgical resectability should be evaluated based on the combination of these imaging findings. Ultrasound is the preferred modality for the initial screening and diagnosis of an abdominal mass. Hepatoblastoma usually presents with a heterogeneous echo signal and significant mass effect on adjacent organs. Ultrasound can confirm the hepatic origin of the tumor by evaluating the movement of the mass with respiration or its vascular supply emanating from the portal vein and hepatic artery[12]. Ultrasound is also very sensitive for detecting portal vein, hepatic vein, and inferior vena cava thrombus[13].Magnetic resonance imaging (MRI) is the preferred cross-sectional imaging modality for the primary tumor because it provides superior soft tissue contrast resolution and does not employ ionizing radiation[13]. On MRI, hepatoblastoma typically has a heterogeneous appearance with a hyperintense signal on T2-weighted images and a hypointense signal on T1-weighted images[13]. The development of hepatocyte-specific MRI contrast provides radiologists with a very useful tool in diagnosing liver tumors[14]. Gadolinium-based compounds are currently used for hepatocyte-specific MRI contrast media. Gadolinium-ethoxybenzyl-diethylenetriamine penta-acetic acid (Gd-EOB-DTPA) has been very useful in the evaluation of pediatric liver lesions, particularly in the sharp distinction of the tumor from normal liver parenchyma, the clear delineation of tumor margins with respect to the biliary tree and blood vessels, and the presence of satellite lesions not otherwise picked up by CT scan. This contrast agent has the potential to improve characterization and staging of hepatoblastoma[15]. Additionally, diffusion-weighted imaging is emerging as a useful sequence for liver lesion detection and characterization. Highly cellular and malignant lesions tend to demonstrate restricted diffusion on this sequence[16]. It can be very useful in detecting and confirming multifocal hepatoblastoma. Vascular invasion is best depicted with gradient-echo imaging or contrast enhanced MR angiography (MRA)[12]. Furthermore, MRA can also detect normal anatomic vascular variations that can guide surgical resection.Since approximately 20% of the newly diagnosed hepatoblastomas present with lung metastasis [17], multi-detector computed tomography (MDCT) lung scanning is required at initial diagnosis and can also be used to scan the abdomen at the same time[13]. Tri-phase imaging is usually recommended for hepatoblastoma which consists of arterial, portal-venous, and hepatic-venous phases. The MDCT appearance of hepatoblastoma is highly variable and depends on the tumor's histologic composition. Although MDCT has fallen from favor over the last decade because of the risks of radiation toxicity and lower detection rate and diagnostic accuracy compared to MRI [18], physicians can justify the use of this scan to reduce the need for anesthesia/sedation[13].Positron emission tomography (PET)/CT has no definitive role in the diagnosis of hepatoblastoma. However, PET/CT can provide valuable information in the assessment of relapsed cases and is especially useful for detecting small early recurrences[19][20][21].The PRETEXT and POST-TEXT SystemThe pre-treatment extent of tumor (PRETEXT) system was developed by SIOPEL to standardize imaging evaluation and risk stratification for hepatoblastoma prior to neoadjuvant chemotherapy[13,[22][23][24], whereas the POST-TEXT (post-treatment extent of disease) system uses the same standards as PRETEXT(Table 1) but classifies hepatoblastoma during neoadjuvant chemotherapy[13,23].
# Introduction
Hepatoblastoma is the most common liver malignancy in children. The incidence is approximately 1.2/1,000,000 and about 100 new cases are diagnosed annually in the United States. Hepatoblastoma accounts for over 90% of the primary hepatic malignancies among children less than 5 years of age. The incidence of hepatoblastoma has increased over the past two decades, partially due to the increased survival of premature and low-birth-weight infants. Hepatoblastoma usually presents with a large abdominal mass and an elevated α-fetoprotein protein (AFP) value, mostly affecting children less than 3 years of age. The current mainstay treatment includes chemotherapy, surgical resection, and transplantation. The advent of platinum-based chemotherapy regimens has dramatically improved the outcomes of hepatoblastoma. Although the Children's Oncology Group (COG), the International Childhood Liver Tumors Strategy Group (SIOPEL), the Society for Pediatric Oncology and Hematology (GPOH), and Japanese Pediatric Liver Tumors Group (JPLT) use different platinum-based chemotherapy regimens, the overall survival in each group is quite similar. Surgery plays a critical role in the management of hepatoblastoma, and complete resection is the only way to achieve cure. Surgical techniques and surgical tools have advanced in past decades which has greatly facilitated precision hepatectomies and metastasectomies. Furthermore, orthotopic liver transplantation provides promising outcomes for those with unresectable hepatoblastoma. The five-year overall survival rate for hepatoblastoma is approximately 80% with those who underwent partial hepatectomy achieving survival rates as high as 91%. Upon review of 292 patients (in 29 separate publications) with hepatoblastoma who underwent liver transplantation, 76% of patients were alive at the time of publication. Forty-one percent of patients with rescue liver transplantation survived, compared with . The pre-treatment extent of tumor (PRETEXT) stage system.
## Stage definition
PRETEXT I Three contiguous hepatic sections are free of tumor PRETEXT II One or two sections have tumor involvement, but two adjoining sections are tumor-free PRETEXT III Two or three sections have tumor involvement, but no two adjoining sections are tumor-free PRETEXT IV All four sections have tumor involvement In addition to these classifications, PRETEXT annotation factors, such as hepatic venous involvement, portal venous involvement, extrahepatic disease, multifocality, rupture, caudate involvement, lymph node metastases, and distant metastases, should also be carefully addressed based on MRI/MDCT findings, according the 2017 PRETEXT guideline. The PRETEXT and POST-TEXT are powerful tools for predicting resectability and survival of hepatoblastoma patients. Detailed and accurate PRETEXT or POST-TEXT staging is of great importance in the surgical management of hepatoblastoma. However, PRETEXT is only approximately 50% accurate compared to pathology assessment with a tendency to over-stage patients using the PRETEXT system. It is recommended that PRETEXT III and IV hepatoblastomas should undergo a central radiology review at diagnosis as well as before surgery.
The Children's Hepatic Tumors International Collaboration-Hepatoblastoma Stratification (CHIC-HS) has been proposed for the newly launched Pediatric Hepatic International Tumor Trial (PHITT) which will prospectively validate this proposed model. Based on the PRETEXT system, age, and AFP levels, CHIC-HS classifies hepatoblastoma into very low-risk, low-risk, intermediate-risk, and high-risk groups.
## Upfront versus delayed surgery
The International Childhood Liver Tumors Strategy Group and Children's Oncology Group approach a newly diagnosed hepatoblastoma with different strategies. The International Childhood Liver Tumors Strategy Group tends to give neoadjuvant chemotherapy to all hepatoblastoma patients and then performs a delayed surgery. The advantage of giving neoadjuvant chemotherapy to all hepatoblastoma patients is that size reduction and down staging of the tumor can be achieved in the majority of cases. Advanced-stage tumors can greatly benefit from neoadjuvant chemotherapy. The SIOPEL trials have revealed that more than half of PRETEXT IV tumors can be completely resected with partial hepatectomy after intensified neoadjuvant chemotherapy. Children's Oncology Group favors up-front resection for initially resectable hepatoblastoma and gives neoadjuvant chemotherapy only to those deemed unresectable in hopes of reducing total chemotherapy administered. For well-differentiated fetal hepatoblastoma, which can be cured with complete surgical resection alone, this approach avoids the need for chemotherapy. Of note, the diagnosis of well-differentiated fetal hepatoblastoma can only be established based on a primary resected sample. It is possible that some of the PRETEXT I and II patients who underwent primary resection may also benefit from reduced cycles of postoperative chemotherapy. Recent PHITT surgical guidelines recommend primary resection only for PRETEXT I and PRETEXT II patients; PRETEXT II tumors should have >1cm radiographic margin from the middle hepatic vein, the retro-hepatic inferior vena cava, and the portal bifurcation. Also, upfront trisectionectomy is no longer recommended for any newly diagnosed hepatoblastoma.
It is equally important to determine the optimal timing of delayed surgery for those who undergo neoadjuvant chemotherapy. The International Childhood Liver Tumors Strategy Group tends to give four cycles of neoadjuvant chemotherapy for standard risk hepatoblastoma. Murphy et al.demonstrated that hepatoblastoma volumes regressed significantly with increasing neoadjuvant chemotherapy cycles. However, tumors often remained anchored to the major hepatic vasculature, showing marginal improvement in resectability criteria. Another study found that after two cycles of neoadjuvant chemotherapy, the majority of stage III and IV hepatoblastomas either did or did not achieve radiographic resectability, and further chemotherapy did not change this outcome. Tumor volume and serum AFP values can be used to assess the responsiveness of chemotherapy. Generally speaking, tumor resectability should be reevaluated after every two cycles of neoadjuvant chemotherapy.
## Advancement in techniques to make tumors resectable
Hepatoblastomas that are unresectable (PRETEXT IV, V+, P+) with standard liver resection should be referred for liver transplantation. Early referral should be conducted in order to achieve the best possible outcome. Trans-catheter arterial chemo-embolization (TACE) alone, or in combination with high-intensity focused ultrasound, may be considered for those with unresectable tumors that are not responsive to primary systemic chemotherapy and are also not suitable for liver transplantations. Furthermore, transarterial radioembolization with yttrium-90 could be considered as adjunctive therapy in unresectable hepatoblastoma and could be used as a bridge to surgical resection or liver transplant.
In the scenario of possible insufficient future liver remnant (FLR), the associating liver partition and portal vein ligation for staged hepatectomy (ALPPS) procedure will be of great usefulness for increasing the volume of FLR. The ALPPS procedure is a two-staged liver resection, combining two established surgical techniques: portal vein ligation and in situ splitting of the liver. During the first stage, the liver is completely divided from the FLR with concomitant portal vein ligation of the lobe that will be removed while preserving ipsilateral arterial blood supply, bile duct, and hepatic vein drainage. The second stage is usually performed 1-2 weeks later with removal of the liver with its portal vein ligated. Wiederkehr et al.reported the initial experiences of ALPPS procedure performed in five pediatric patients; a rapid growth of the remnant livers was observed in all but one patient. The increase in the ratio of FLR to total liver volume ranged from 62% to 102% in four patients. The only postoperative complication was an asymptomatic right pleural effusion that was aspirated during the second stage procedure. The ALPPS has also been successfully performed in a 54 day old infant with hepatoblastoma. However, since as little as 20-25% of FLR is sufficient for pediatric patients undergoing liver resection, ALPPS should only be reserved for those high-risk patients who would otherwise not be a liver transplant candidate. Additionally, portal vein embolization alone is another option to induce hypertrophy of FLR.
## Advances intraoperative techniques and approaches to local control
In recent years, new technological advances have been employed as adjuncts to pediatric hepatic resections with exciting results. These include the use of intraoperative ultrasound, the use of image guided three-dimensional reconstruction, and the use of indocyanine green (ICG) during liver resection and metastatic lesion resection. Newer approaches to local control surgery have been to use laparoscopic liver resection and more extreme resection techniques.
Intraoperative ultrasound (IOUS) during hepatic resection in adults was first described with good results as early as the 1970s. The use of IOUS as an adjunct to adult liver resections in cases of both hepatic metastasis from gastrointestinal malignancies as well as for defining the proper hepatic transection planes is well reported with good results. The use of IOUS has continued to lead to changes in operative strategy despite well-reported advances in preoperative imaging. While the use of IOUS in pediatric liver resections has been reported as early as 1999, most reports were of only a few patients from single center institutions. In 2015, Felsted et al.described a study examining the use of IOUS in pediatric patients undergoing liver resection and found that, even with the advances in preoperative MRI imaging, discordant findings were found in approximately 20% of patients intraoperatively with ultrasound that changed the operative management in 14% of cases. While it is established that MRI is the best choice for preoperative imaging of pediatric hepatic malignancies with sensitivity of 1-3 mm, there are cases in which MRI is limited. In cases of complex or distorted hepatic vascular anatomy, the use of IOUS can be used to better assess the relationship of the tumor to the corresponding hepatic veins. Of note, particularly challenging in preoperative imaging is that tumors that occupy segment IV can be distorted secondary to the umbilical fissure, unclear middle vein trajectory, and sometimes obliterated left portal vein secondary to tumor involvement. The images of preoperative MRI might also further be distorted secondary to parenchymal compression from local tumor effects as well as motion artifacts during the time of imaging. With IOUS, a spatial resolution of 3-5 mm can be obtained. Most importantly, the tissue is able to be moved as needed in order to best visualize important structures and determine relationships in an effort to achieve a proper transection plane in a parenchyma sparing resection. Ultrasound should be readily available to the surgeon to ensure the correct plane of parenchymal transection and preservation of feeding and draining vessels to the remnant liver.
Technological advances have now created 3D image-processing software dedicated to virtual simulation in liver surgery. Such techniques provide not only 3D reconstructions of the liver structures, but also produce individualized anatomical information of the tumors and vascular structure. Surgeons can virtually perform liver resections under realistic anatomic conditions, evaluating the impact of the resection on the blood supply and the drainage of the liver remnant. Three-dimensional tumor visualization and virtual simulation of tumor resections provides the basis to successfully plan for the treatment of a complex tumor.
Cancers 2019, 5 of 14 unclear middle vein trajectory, and sometimes obliterated left portal vein secondary to tumor involvement. The images of preoperative MRI might also further be distorted secondary to parenchymal compression from local tumor effects as well as motion artifacts during the time of imaging. With IOUS, a spatial resolution of 3-5 mm can be obtained. Most importantly, the tissue is able to be moved as needed in order to best visualize important structures and determine relationships in an effort to achieve a proper transection plane in a parenchyma sparing resection. Ultrasound should be readily available to the surgeon to ensure the correct plane of parenchymal transection and preservation of feeding and draining vessels to the remnant liver. Technological advances have now created 3D image-processing software dedicated to virtual simulation in liver surgery. Such techniques provide not only 3D reconstructions of the liver structures, but also produce individualized anatomical information of the tumors and vascular structure. Surgeons can virtually perform liver resections under realistic anatomic conditions, evaluating the impact of the resection on the blood supply and the drainage of the liver remnant. Three-dimensional tumor visualization and virtual simulation of tumor resections provides the basis to successfully plan for the treatment of a complex tumor. Virtual resections can substantially contribute to surgical strategies for children with complex hepatic tumors. This tool may also determine which specific surgical procedure is required such as an extended liver resection instead of primary transplantation in certain conditions. Virtual resections can substantially contribute to surgical strategies for children with complex hepatic tumors. This tool may also determine which specific surgical procedure is required such as an extended liver resection instead of primary transplantation in certain conditions. Additionally, three-dimensional printing is taking a step forward by producing a real, tactile disease model. Three-dimensional printing models may help in planning surgical resection and even guide real-time resection. These models provide a practical, hands-on tool that has a number of unique applications for surgical planning. This model accurately mimics the liver structure and has been demonstrated to be very useful in adult liver resection and liver transplantation.
Another adjunct for pediatric hepatic resection is the use of ICG imaging. The use of preoperatively injected intravenous ICG and near infrared imaging equipment has emerged as a novel therapy in cases of solid organ malignancy and metastatic resections. The use of ICG imaging emerges from its ability to be taken up by normally functioning hepatocytes and then undergo a period of post-injection hepatic washout. The degree of hepatic washout has been proposed as a mechanism for predicting post-operative liver dysfunction in adults. Protein-bound ICG fluoresces at a specific wavelength that is able to be picked up by specially designed intraoperative imaging equipment using near infrared laser technology. Using this specialized equipment, which was initially reported for adult biliary surgery for better visualization to avoid ductal injury, sites of hepatic primary malignancy and metastatic locations are able to be detected with green fluorescence. In addition to the identification of small and difficult to palpate lymph nodes and metastasis, the use of ICG can also be used to identify small, superficially located hepatic tumors. This modality can be used to help achieve a negative resection status, safely remove tumors from closely underlying vascular structures, as well as assess the degree of any remaining malignancy. Because of these advances, ICG has started to gain popularity in oncologic surgery. Indocyanine green can be used for imaging both primary liver cancers and metastases. Healthy liver tissue clears ICG within a few hours, whereas liver tumor tissue retains ICG. The current literature studying ICG in liver surgery uses selective portal vein and hepatic vein injections to highlight segmental anatomy as a guide for anatomical liver resection and for identifying small satellite lesions. The use of ICG is well reported internationally in the resection of hepatoblastoma. Generally, ICG (0.5-1 mg/kg) is intravenously injected 48-72 h prior to surgery to ensure hepatic clearance. This method aids in determining the resection line and identifying residual tumors.
Laparoscopic liver resection is becoming increasingly popular for adult liver resection. In experienced hands, laparoscopic liver resections are safe with acceptable morbidity and mortality for major hepatic resections. Laparoscopic surgery provides patients with smaller incisions, less blood loss, and shorter hospital stays. Laparoscopic ultrasound is crucial, and a thorough knowledge of both B-mode and Doppler ultrasonography is mandatory for accurate laparoscopic liver resections. Use of on-demand intermittent inflow occlusion and low central venous pressure anesthesia are very effective for limiting blood loss during laparoscopic liver resection. Advanced technologies, such as energy devices and staplers, are required for laparoscopic operations. The energy devices are effective for transecting the superficial liver parenchyma. For dissection of deeper parenchyma, prior identification and selective hemostasis of larger vessels by the use of combination of cavitron ultrasonic surgical aspirator (CUSA) and energy devices and staplers is recommended. Some pediatric surgeons have performed laparoscopic liver resection for selective childhood liver tumors, including hepatoblastoma, but limited space in the abdominal cavity remains a major obstacle for childhood laparoscopic liver resection. However, hepatoblastoma usually significantly shrinks after neoadjuvant chemotherapy and is usually not associated with hepatic cirrhosis. These may help to facilitate a laparoscopic liver resection. When considering laparoscopic resection of hepatoblastoma, appropriate tumor and patient selection is undoubtedly the key to success.
Extreme liver resection for advanced-stage (POST-TEXT III and IV) hepatoblastoma seems to have comparable overall survival when combined with chemotherapy as compared to liver transplantation. Two major studies reporting these results presented patients with positive microscopic resection margins without local recurrence. The application of intensive neoadjuvant chemotherapy regimens increases the possibility of extensive liver resection. In patients with an initial PRETEXT-IV tumor as the only high-risk feature, half of the tumor can be completely resected with partial hepatectomy after intensive neoadjuvant chemotherapy. Joerg Fuchs et al.reported extensive liver resection in a series of 27 cases of POST-TEXT III and IV hepatoblastoma, with a five-year overall survival of 80.7%. El-Gendi A et al.reported a three-year overall survival in 86.6% of a series of 15 cases of POST-TEXT III and IV hepatoblastoma patients who underwent extensive liver resection. Recent evidence suggested that, in the context of cisplatin-based chemotherapy, the presence of microscopically positive resection margin did not influence the outcome. In the SIOPEL study, with a median follow-up of 67 months, local relapse occurred in 3/58 (5%) patients with microscopically positive resection margin and in 23/371 (6%) patients with complete resection. The 5 year overall and event-free survival was 91% and 86%, respectively, for the microscopically positive resection margin group and 92% and 85%, respectively, for the complete resection group. This renders further support for extensive liver resection and may increase the chances of patients with POST-TEXT III and IV to undergo extensive liver resection. This aggressive surgical resection may mitigate the need for orthotopic liver transplantation in selective advanced cases. However, preparation for backup liver transplantation should always be considered.
## Resection of lung metastasis
The lung is the most common metastatic site for hepatoblastoma and approximately 20% of the initially diagnosed hepatoblastoma cases have also presented with lung metastasis. The initial treatment for hepatoblastoma with lung metastasis is neoadjuvant chemotherapy. Among the patients with initial lung metastases, more than half achieved complete remission of the lung lesions with intensive neoadjuvant chemotherapy. Metastasectomy for residual pulmonary nodules after neoadjuvant chemotherapy should be aggressively performed. Wanaguru et al.reported that aggressive surgical resection of lung metastasis achieved long-term cure in eight hepatoblastoma cases in the context of chemotherapy. The only absolute contradictions for metastasectomy would be insufficient pulmonary function. Moreover, lung metastasis should always be resected prior to liver transplantation.
Lung metastasis can be safely resected with traditional thoracotomy or thoracoscopic surgery. The traditional thoracotomy provides the ability to manually palpate the lungs, which was once considered essential in identifying lung metastasis. For video-assisted thoracoscopic surgery, CT-guided lung nodule localization using the combined techniques of methylene blue blood patch and hook wire is safe, technically feasible, and successful in children. Using this combination of techniques will consistently yield a pathological diagnosis.
Intraoperative ICG fluorescence imaging is feasible and useful for identifying small viable metastatic lung lesions. Kitagawa et al.reported that ICG can detect lung lesions as small as 0.062 mm in diameter, and all of the pathologically positive lesions were clearly fluorescence positive in a study of 10 patients. Most researchers suggested an interval between the administration of ICG (0.5 mg/kg) and pediatric surgery of at least 1-4 days to decrease the background fluorescence. Navigation surgery using ICG could not detect the tumors located at a depth of more than 10 mm from the organ surface. metastatic lung lesions. Kitagawa et al.reported that ICG can detect lung lesions as small as 0.062 mm in diameter, and all of the pathologically positive lesions were clearly fluorescence positive in a study of 10 patients. Most researchers suggested an interval between the administration of ICG (0.5 mg/kg) and pediatric surgery of at least 1-4 days to decrease the background fluorescence. Navigation surgery using ICG could not detect the tumors located at a depth of more than 10 mm from the organ surface.
## Management of disease relapse
Relapsed hepatoblastoma occurs in less than 12% of patients with complete remission after the first line of treatment. Most of the relapses happen in the liver and lungs. Combined treatment with chemotherapy and surgical removal is essential for long-term survival. All four patients in the JPLT-1 study with local relapse were salvaged with chemotherapy and surgical resection. In the SIOPEL series, 52% (31 of 59) of the relapsed patients achieved a second complete remission. Although surgical resection of a local relapse may be difficult after a previous surgery, complete surgical resection was achieved in 15 out of 21 patients who experienced local relapse in the SIOPEL study. Indocyanine green may offer accurate guidance in the resection of the relapsed disease because it enables the identification of small viable lesions during surgery that otherwise may not be defined by cross sectional imaging. It can be used not only for the guidance of a second resection for the local relapse but also for the clearance of any residual pulmonary nodules in preparation for orthotopic liver transplantation.
Orthotopic liver transplantation is an option for those with unresectable local relapse. However, Otte et al.reported that children who undergo rescue liver transplantation have a considerably worse prognosis compared with those who undergo primary liver transplantation. The six-year post-transplantation overall survival was 82% for 106 patients who received a primary orthotopic liver transplantation but only 30% for 41 patients who underwent a rescued orthotopic liver transplantation. A moderate increased success rate was reported by Trobaugh-Lotrariothrough summarizing data in 29 separate publications. Of 41 patients with rescue liver transplantation after initial attempt at resection, seventeen (41%) were alive. Encouragingly, Sakamoto et al.reported a 78% of three-year recurrence-free survival in a cohort of 15 patients with a rescued orthotopic liver transplantation. Percutaneous ablation therapy and transarterial radioembolization using yttrium-90 microsphere might be an effective alternative for the control of unresectable local relapse in patients after liver transplantation.
Lung relapse can be solitary or multiple, unilateral or bilateral. The value of pulmonary metastasectomy for lung relapse is not as well-established as the management of residual lesions after neoadjuvant chemotherapy. Meyers et al.reported that only 4 of the 13 patients with lung relapse who underwent thoracotomy were long-term survivors. Of note, five of them only had a thoracotomy and biopsy. Later data suggests that surgery and combined chemotherapy should be offered to all patients with lung relapse. In a SIOPEL study of 59 cases relapsed after achieving complete remission, 31 patients (52%) achieved a second complete remission. Of the twenty-seven patients that had lung relapses, 15 could be resected to achieve a second remission. Shi et al.reported surgical experiences of 10 patients with lung relapse, one with bilateral lung metastasis. Eight were effectively treated with pulmonary metastasectomy, which provided long term survival. The other two succumbed to extrapulmonary metastasis. Multiple repeat thoracotomies can be performed to clear pulmonary recurrences as needed in order to increase the disease-free interval.
# Conclusions
The PRETEXT system and platinum-based chemotherapy have laid out a foundation for the current management of hepatoblastoma. Substantial progress has been made in the surgical arena of hepatoblastoma. Other than the promising outcomes of transplantation, the most exciting part of the surgical management of hepatoblastoma may be the introduction of ICG-guided and 3-D model guided surgery. These new technologies have led to precise resection of hepatic tumors that may not otherwise be resectable and can be effective for the treatment of primary or recurrent lung metastases. Furthermore, ALLPS further pushes the envelope for the success of surgical resection by increasing the volume of potentially insufficient future liver remnants. All of these advancements will enable surgeons to provide better surgical outcomes for hepatoblastoma. |
Selective C–C Coupling Reaction of Dimethylphenol to Tetramethyldiphenoquinone Using Molecular Oxygen Catalyzed by Cu Complexes Immobilized in Nanospaces of Structurally-Ordered Materials
## Preparation of cu 2+ (mono)-magadiite
Cu 2+ (mono)-magadiite was prepared in the same manner as reported by Kim et al. [bib_ref] Intracrystalline and electronic structure of copper(II) complexes stabilized in two-dimensional aliminosilicate, Choy [/bib_ref]. A Cu mononuclear complex Cu(ethylenediamine)2(ClO4)2 was synthesized by addition of ethylenediamine (1.3 mL, 20 mmol) to the MeOH solution containing Cu(ClO4)2·6H2O (10 mmol), and the resulting solution was refluxed for 4 h. After the reaction, the solution was cooled to room temperature to precipitate the purple solid. The precipitate was filtered and washed with ethanol to afford Cu(ethylenediamine)2(ClO4)2. Next, Cu(ethylenediamine)2(ClO4)2 (100 µmol) was dissolved in EtOH (10 mL), and magadiite (0.4 g) was added into the above solution. The resulting mixture was further stirred for 6 h at 313 K. After the reaction, the obtained solid was filtered, washed with EtOH (100 mL), and dried to afford a light purple powder.
## Reuse experiment of g 4 -cu 2+ 12
1a (0.5 mmol) was reacted using fresh G4-Cu 2+ 12 (Cu: 5 µmol) in 4 mL α,α,α-trifluorotoluene (TFT), after which the synthesized 2a was removed from the reaction mixture by filtration. 1a (0.5 mmol) was added to the filtered TFT solution, still containing the original G4-Cu 2+ 12, and the mixture was vigorously stirred at 323 K for 18 h. By repeating this process, it was determined that the G4-Cu 2+ 12 catalyst could be reused without significant loss of its activity and selectivity; the yield of 2a from the first reaction, as determined by 1 H NMR, was 96%, while the isolated yields were 83% (fresh catalyst), and 93% (first reuse).
## S2 esr measurement
The spectra were recorded at the Xband using a Bruker EMX-10/12 spectrometer with a 100 kHz magnetic field modulation at a microwave power level of 10.0 mW. G4-Cu 2+ n (n = 2, 8, 12, 16, and 24; Cu: 0.5 µmol) in CHCl3 (0.1 mL) was placed in a quartz ESR tube under an Ar atmosphere, and measured at 298 K. In the case of the heterogeneous Cu catalysts, the Cu catalyst (Cu: 0.5 µmol) was introduced in a quartz ESR tube, evacuated at room temperature, and subjected to analysis at 298 K.
## Xafs measurement
The Cu K-edge XAFS spectrum of G4-Cu 2+ 12 was recorded in transmission mode at room temperature. Fourier transforms of k 3 -weighted EXAFS spectra were performed in the 4 Å < k < 12 Å range to obtain radial structural functions. Curve-fitting analysis was performed with the inverse FT of the 1.2 Å < R < 2.4 Å range. The coordination numbers (CN) and interatomic distances (R) were estimated by curve-fitting analysis using Cu-Cl and Cu-N shell parameters obtained from reference samples of CuCl2 [bib_ref] The crystal structure of anhydrous cupric chloride, and the stereochemistry of the..., Wells [/bib_ref] and Cu(ImH)4SO4 [bib_ref] Metal complexes with mixed ligands. 4. The crystal structure of tetrakisimidazole Cu(II)..., Fransson [/bib_ref] , respectively. In the case of Cu 2+ -magadiite, the spectrum was recorded at 10 K using a cryostat. Curve-fitting analysis was conducted with the inverse FT of the 1.0 Å < R < 3.3 Å range, and the CN and R were estimated by curve-fitting analysis using Cu-N/O and Cu-Cu shell parameters obtained from a reference sample of [Cu(OH)TMEDA]2Cl2 [bib_ref] Multitechnique approach to reveal the mechanism of copper(II)-catalyzed arylation reactions, Tromp [/bib_ref].
## Product identification
The reaction products were identified by 1 H and 13 C NMR and, in each case, the chemical shifts of the products were in agreement with those reported in the literature, as summarized below. 3,3'-Di-tert-butyl-5,5'-dimethyldiphenoquinone (2c) (CAS-RN 2417-00-1) [bib_ref] Efficient oxidative coupling of 2,6-disubstituted phenol catalyzed by a dicopper(II) complex, Liao [/bib_ref] (Likely a mixture of cis and trans isomers [bib_ref] Notiz zur rotationsisomerie bei p-diphenochinonen, Rieker [/bib_ref]. . Simultaneous in situ UV-vis spectra recorded during G4-Cu 2+ 12-catalyzed oxidative coupling of DMP under an Ar atmosphere as shown in Scheme S1.
# Scheme and figures
[fig] 3, 3 ': 5,5'-Tetramethyldiphenoquinone (DPQ, 2a) (CAS-RN 4906-22-3) [5] 3,3',5,5'-Tetramethylbiphenyl-2,2'-diol (TMBP, 3a) (CAS-RN 2417-04-1) [5] Poly(2,6-dimethylphenylene ether) (PPE, 4a) (CAS-RN 42397-25-1) [6] 3,3',5,5'-Tetraisopropyldiphenoquinone (2b) (CAS-RN 2178-51-0) [5] [/fig]
[fig] Figure S1, Figure S2, Figure S3: UV spectra of Cu 2+ -magadiite and magadiite. UV-vis spectrum of LMCT band of Cu 2+ -magadiite. Cu K-edge XANES spectra of (a) Cu 2+ -magadiite, (b) Cu 2+ -magadiite (used), (c) [Cu(OH)TMEDA]2Cl2, (d) Cu 2+ (mono)-magadiite, (e) CuO, and (f) Cu2O. [/fig]
|
Fatal Case of Mediterranean Spotted Fever Associated with Septic Shock, Iran
[bib_ref] Epidemiological evaluation of Mediterranean spotted fever in children of the Karak province..., Nafi [/bib_ref] [bib_ref] Severe clinical forms of Mediterranean spotted fever: a case series from an..., Baymakova [/bib_ref] [bib_ref] Cases of Mediterranean spotted fever in southeast of Iran, Farrokhnia [/bib_ref] [bib_ref] The pathology of fatal Mediterranean spotted fever, Walker [/bib_ref] [bib_ref] The pathology of fatal Mediterranean spotted fever, Walker [/bib_ref] [bib_ref] Two cases of Israeli spotted fever with Purpura fulminans, Sharon District, Cohen [/bib_ref] [bib_ref] Mediterranean spotted fever as a cause of septic shock, Abdeljelil [/bib_ref]
[fig] Figure: Phylogenetic analysis of Rickettsia species from a patient with Mediterranean spotted fever associated with septic shock, Iran (in bold), confirming infection with R. conorii subspecies israelensis. A) gltA gene; B) 17KD gene; C) ompA gene. Tree was constructed with the maximum-likelihood method algorithm (Tamura-Nei model). The test was performed with bootstrap (500 repetitions) by MEGA X 10.1 software (https://www.megasoftware.com).RESEARCH LETTERSTo determine Bartonella spp. dynamics, we sampled bats and bat flies across 15 roosts in Costa Rica. PCR indicated prevalence of 10.7% in bats and 29.0% in ectoparasite pools. Phylogenetic analysis of 8 sequences from bats and 5 from bat fly pools revealed 11 distinct genetic variants, including 2 potentially new genotypes. B artonella, the causative agent of bartonellosis, is a genus of gram-negative bacteria. Bartonellosis causes a range of symptoms from severe to life-threatening (e.g., endocarditis and meningitis). Clinical syndromes from Bartonella infections include trench fever (B. quintana), cat scratch disease (B. henselae), and Carrion's disease (B. bacilliformis)(1). Bats (Order Chiroptera) and their blood-1 These authors contributed equally to this article. [/fig]
[table] Table: Laboratory findings in a patient with Mediterranean spotted fever associated with septic shock, Iran* [/table]
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Effect of Levan Supplement in Orange Juice on Weight, Gastrointestinal Symptoms and Metabolic Profile of Healthy Subjects: Results of an 8-Week Clinical Trial
Levan is a commonly used dietary fiber of the fructans group. Its impact on health remains undetermined. This double blind controlled study aimed to investigate the effect of 8 weeks' daily consumption of 500 mL of natural orange juice enriched with 11.25 g of levan compared to the same amount of natural orange juice without levan on weight, gastrointestinal symptoms and metabolic profiles of 48 healthy volunteers. The statistical analyses compared between-and within-group findings at baseline, 4 weeks and study closure. The compared parameters were: weight, blood pressure, blood laboratory tests, daily number of defecations, scores of stool consistency, abdominal pain, bloating, gas, dyspepsia, vomiting and heartburn. Despite a higher fiber level recorded in the study group, there was no significant difference in the effect of the two kinds of juices on the studied parameters. Both juices decreased systolic and diastolic pressures, increased sodium level (within normal range), stool number, and bloating scores, and decreased gas scores. In conclusion, levan itself had no effect on weight, gastrointestinal symptoms or metabolic profile of healthy volunteers. Its possible effect on obese, hypertensive or hyperlipidemic patients should be investigated in further studies.
# Introduction
Levan belongs to a group of fructans, which are biopolymers of fructose and composed of fructofuranosyl residues joined by β(2,6) and β [bib_ref] Prebiotic properties of Levan in rats, Jang [/bib_ref] linkages. There are two main natural fructans, inulin and levan, which are distinguished by the type of linkage. Levan can be found in plants and in the bio-products of microorganisms. The length of levan varies from ten to thousands of fructose residues [bib_ref] Microbial levan, Han [/bib_ref]. One advantage of fructose polymers is their ability to be fermented by gut microflora which, in turn, improves the intestinal flora [bib_ref] Dietary modulation of the human microbiota. Introducing the concept of prebiotics, Gibson [/bib_ref] and increases mineral absorption [bib_ref] A combination of prebiotic short-and long-chain inulin-type fructans enhances calcium absorption and..., Abrams [/bib_ref] [bib_ref] Effects of fructooligosaccharides on the absorption of iron, calcium and magnesium in..., Ohta [/bib_ref]. Prebiotic fibers, such as inulin, pass through the small intestine without being digested and are fermented in the large intestine. Dietary inulin and other fructo-oligisaccharides have been shown to markedly increase bifidobacterium and lactobacillus in healthy humans and animals [bib_ref] The bifidogenic nature of chicory inulin and its hydrolysis products, Roberfroid [/bib_ref]. The prebiotic nature of levan is less understood. Several groups have studied the prebiotic effects of levan in a rat model and showed that total fecal weight, short-chain fatty acid production and the total number of microorganisms and acid-producing bacteria increased significantly with its use [bib_ref] Prebiotic properties of Levan in rats, Jang [/bib_ref]. The hypocholesterolemic effect of levan was also studied in a rat model and was found to have a positive effect on weight and plasma lipids [bib_ref] In vitro digestibility and fermentability of levan and its hypocholesterolemic effects in..., Yamamoto [/bib_ref]. Finally, levan was shown to have an immunostimulatory effect, as demonstrated by the induction of IL-12 production and suppression of T-helper type 2 response and IgE production [bib_ref] Levan, a major fraction of fermented soybean mucilage, displays immunostimulating properties via..., Xu [/bib_ref]. The effect of Levan on humans was largely not investigated in controlled studies till now. The only controlled study regarding the metabolic effect of Levan was conducted by in 2003 on 29 Korean women [bib_ref] The effect of fructose polymer levan on the body fat accumulation and..., Kang [/bib_ref]. This study demonstrated that an addition of 6 g of Levan was effective in controlling weight, body fat, cholesterol profile and triglyceride levels.
Levan is naturally present in various food products and regularly consumed by humans. Its content in food can be increased by applying fermentation techniques. "Natto" is a traditional Japanese food made of fermented soybeans, which contains high concentrations of levan that are produced during the fermentation process. Its concentration can also be increased by the enzyme, levansucrase. In juices, levansucrase can hydrolyze naturally occurring sucrose into glucose and fructose, and polymerize the obtained fructose into levan. By means of these processes, the amount of sucrose in a fruit juice can be reduced and the amount of levan (a dietary fiber) can be increased. These changes are very important for the dietary industry, especially in an era of functional food.
Natural orange juice has a very high percent of sucrose as a natural component. The aim of this study was to compare the effect of 8 weeks of administration of natural orange juice fermented with levansucrase (reduced sugar percentage and increased fiber content) with that of regular natural orange juice on the weight, gastrointestinal symptoms and metabolic profile of healthy volunteers.
# Materials and methods
This was a double blind parallel controlled study of healthy volunteers. It was conducted in the Unit of Clinical Nutrition of Tel-Aviv Sourasky Medical Center, Israel (Registered under ClinicalTrials.gov Identifier no. NCT00123456). The study design was approved by the local Helsinki Committee and all participants gave written informed consent. The subjects were recruited by newspaper advertisements and received financial compensation. They all underwent an initial screening evaluation, which included: medical history, medical examination, weight and blood pressure measurements, a 3-day food diary in order to establish their dietary habits, baseline stool and gastrointestinal questionnaires, blood tests for glucose, HbA1C, urea and creatinine, uric acid, GOT, GPT, GGT, alkaline phosphatase, total cholesterol, LDL and HDL cholesterol, triglycerides, total bilirubin, iron, transferrin, ferritin, sodium, potassium, chloride, calcium, magnesium, inorganic phosphate, zinc ,albumin, TSH and complete blood count.
The inclusion criteria were: healthy volunteers aged 18-60 years, written informed consent, ability to drink orange juice and regular eating patterns. The exclusion criteria were: chronic disease (diabetes, cancer, chronic obstructive pulmonary disease, metabolic syndrome, overweight, kidney failure, heart disease and osteoporosis), antibiotic treatment during the past 4 weeks, taking regular probiotic or prebiotic supplementation (unless a 2-week pre-study washout is carried out), intestinal disorders (inflammatory bowel disease, irritable bowel disease, celiac, colon cancer), intestinal surgery, use of medications for lowering cholesterol and/or sugar control, pregnancy, abnormal baseline blood test results (liver and/or kidney function, total cholesterol >250 mg/dL and fasting glucose >120 mg/dL).
The consecutive enrollees were divided into two groups by double blind randomization (1:1.5). Those in the control group were assigned to drink 500 cc of regular fresh pasteurized orange juice daily for 2 months, while those in the study group drank the same amount of orange juice that was reduced in sugar and contained 11.25 g of levan [fig_ref] Table 1: Comparison between regular juice and levan-enriched juice [/fig_ref]. Specifically, the latter juice contained 5 g/L of sucrose compared with 44 g/L in regular juice, and a higher glucose and sodium content (39 versus 21 g/L and 53 versus 9 mg/mL, respectively) produced during the fermentation. Both juices were produced by Gan Shmuel Factory, Foods, Ltd., Israel. Both juices came in the same packings, same bottles (but labeled as A or B), have the same texture and a similar taste. All participants were asked to adhere to their regular diet. Because of levan consumption, the study subjects consumed 11.25 g of added fibers to their previous diet. All participants were asked to fill out a daily stool and gastrointestinal status questionnaire throughout the study period. The following items were included: compliance to juice consumption, number of defecations, and stool consistency and color. They were requested to score the intensity of abdominal pain, bloating, gas, dyspepsia, and report the presence of vomiting or heartburn. They were also asked to attend the Unit of Clinical Nutrition every two weeks for weight and blood pressure measurements, medical check-ups and interviews about compliance, general feeling and adverse events. Blood was drawn in the middle and the end of the study period (week 4 and week 8), and the above-cited tests were run. A telephonic interview was conducted weekly. At study closure, the subjects filled in a satisfaction questionnaire regarding the juice they had drunk as well as a 3-day food diary to ensure that there had been no changes in their regular diets.
In addition to the study and control groups, five volunteers were asked to drink standard and levan-supplemented juice with an interval of several days in between juices in order to determine the glycemic index of each juice. They drank the specified juice after an overnight fast of 12 h, and blood was drawn for insulin and glucose at intervals of 0, 30, 60, 90 and 120 min after juice consumption. The areas under the glucose and the insulin curves were calculated and compared. Each subject was his/her own control.
Selected parameters were analyzed statistically and compared between the study and control groups and within each group at the beginning, middle and end of the 8-week study period. The parameters were: body mass index (BMI), systolic and diastolic blood pressure, blood laboratory tests (chemistry, complete blood count, liver enzymes, lipid profile, HBA1C, iron parameters, TSH), as well as daily number of defecations, scores of stool consistency, and the presence and severity of any existing abdominal pain, bloating, gas, dyspepsia, vomiting and heartburn. The data of the study group were analyzed by one-way analysis of variance with repeated measures (points in time) using the mixed model. Pair-wise comparisons between time points were performed using Hochberg's correction whenever the effect of time was found significant. When the interaction between study group and time was significant, it was sliced to test for simple effects for each group and time point. Values of p < 0.05 were considered statistically significant. In order to compare these parameters between the study and control groups and to examine changes over time, the data were first averaged over each week and then grouped into three time intervals, i.e., week one (baseline), weeks 3-6, and weeks 7-8. Comparisons between areas under the glucose and insulin curves for evaluation of the glycemic index were performed using the paired two-tailed t-test. Each participant was used as his/her own control. All statistical analyses were performed using SAS for windows 9.2.
# Results
Twenty-four of the 74 volunteers were not enrolled at the time of recruitment: 6 were not interested after hearing the details of the study, 15 had abnormal blood tests, one had a chronic disease, one does not like drink juice and one did not have regular eating habits. Another two subjects were dropped shortly after study entry due to non-compliance, leaving a total of 48 subjects. They were randomly divided into two groups: 19 drank 500 cc of regular fresh pasteurized orange juice every day for 8 weeks (controls) and 29 drank sugar-reduced orange juice that contained 11.25 g of levan. The demographics of the study population are presented in [fig_ref] Table 2: Demographics of the study population [/fig_ref]. The self-reported baseline fiber consumption in both groups was similar: 11.2 g in the study group versus 13 g in the control group (p = 0.135). On baseline the participants did not drink fruit juice on a daily basis but occasionally only. Three-day food diary at the closure of the study confirmed that the participants did not perform changes in the regular diet (as they were asked not to do). The results of the mixed model for relative changes in the gastrointestinal status during the 8-week study period are presented in [fig_ref] Table 3: Results of mixed model for relative changes in gastrointestinal symptoms during 8... [/fig_ref]. Drinking orange juice caused a significant increase in the number of stools per week in both groups (p = 0.02) on expense of an increase in semi-solid and fluid stools (p = 0.023 and 0.01, respectively). There was no difference in the number of stools per week between the two groups (p = 0.79). In addition, consumption of orange juice increased the number of complaints of abdominal bloating and decreased the gas scores (p = 0.05 and 0.01, respectively) in both control and study groups (p = 0.39 and 0.93, respectively). There were no between-or within-group differences over time with regard to other gastrointestinal symptoms (abdominal pain, vomiting, heartburn and epigastric pain).
The drinking of regular or levan-enriched sugar-reduced juice over 8 weeks produced minimal if any effects on metabolic profiles and blood tests [fig_ref] Table 4: Results of mixed model for relative changes in BMI, blood pressure and... [/fig_ref]. BMI, glucose, HbA1C, cholesterol (both LDL and HDL), and triglyceride levels underwent no between-or within-group changes. Both systolic and diastolic pressures decreased significantly after the regimen of orange juice drinking was initiated (p = 0.04 and 0.03, respectively), but with no differences between the two groups (p = 0.27 and 0.67, respectively). Both the baseline and the endpoint blood pressure were normal in both groups. The sodium level increased significantly during the study in both groups (p = 0.009), but with no difference between the groups (p = 0.38), and still remaining within the normal range. Statistical analysis showed transient changes in creatinine and bilirubin levels (within normal limits), but both levels returned at week 8 to the baseline levels. Calcium levels decreased significantly at week 4 (within normal limits) compared to baseline in both groups, and returned to original levels at week 8 (p = 0.0005) for both groups (p = 0.91). The rest of the blood tests were not influenced by the consumption of either type of orange juice, nor were there any adverse effects.
The glycemic index of both types of juices was examined in five volunteers [fig_ref] Table 5: Glycemic index of levan-enriched and regular juice [/fig_ref]. The area under the insulin curve tended to be lower in the levan-enriched group compared to the regular juice (p = 0.078). The area under the glucose curve was similar in both groups (p = 0.95), i.e., the glycemic index of levan-enriched juice was lower than that of the regular one.
# Discussion
The results of the double-blind controlled study on healthy volunteers of 8 weeks administration of natural orange juice fermented with levansucrase (reduced sugar percentage and increased fiber content) versus regular natural orange juice showed no significant impact of levan on weight, gastrointestinal symptoms and metabolic profile.
Levan-containing orange juice also caused no adverse events. Both regular and sucrose-reduced levan-enriched orange juices, however, decreased systolic and diastolic pressures, increased sodium levels (within normal range), increased stool number, and bloating scores, and decreased gas scores. This indicates that these effects were induced by the orange juice itself and not by the reduction of sugar or the increase in fiber content. The participants in the study group followed an 8-week diet that was almost double the fiber content than that of the controls (22.45 g versus 13 g) and than their pre-study diet (22.45 g versus 11.2 g). This change in fiber content had no effect on either the metabolic profile or gastrointestinal parameters.
Many studies that emerged during the past decades described a benefit of dietary fiber intake, such as a decreased risk of colorectal cancer, [bib_ref] Dietary fiber and colorectal cancer risk: A nested case-control study using food..., Dahm [/bib_ref] lowering of cholesterol and triglycerides levels [bib_ref] The effects of barley-derived soluble fiber on serum lipids, Talati [/bib_ref] , and relief of irritable bowel syndrome symptoms [bib_ref] Effect of fibre, antispasmodics, and peppermint oil in the treatment of irritable..., Ford [/bib_ref]. Fibers which can be fermented by intestinal flora (prebiotics) are of special interest. Inulin, which, like levan, is a member of the fructans family, has a well-known prebiotic effect [bib_ref] Dietary modulation of the human microbiota. Introducing the concept of prebiotics, Gibson [/bib_ref] [bib_ref] The bifidogenic nature of chicory inulin and its hydrolysis products, Roberfroid [/bib_ref] [bib_ref] The bifidogenic effect of inulin and oligofructose and its consequences for gut..., Meyer [/bib_ref]. It has a proven hypo-triglyceridemic effect [bib_ref] The bifidogenic effect of inulin and oligofructose and its consequences for gut..., Meyer [/bib_ref] [bib_ref] Systematic review of the effects of inulin-type fructans on blood lipid profiles:..., Wu [/bib_ref] [bib_ref] Dietary fructans and serum triacylglycerols: A meta-analysis of randomized controlled trials, Brighenti [/bib_ref] , an ability to enhance mineral absorption [bib_ref] A combination of prebiotic short-and long-chain inulin-type fructans enhances calcium absorption and..., Abrams [/bib_ref] , and even a possible effect of reducing the risk of colon cancer [bib_ref] Dietary fiber and colorectal cancer risk: A nested case-control study using food..., Dahm [/bib_ref]. Unlike inulin, levan has not been investigated in depth. Some in vitro studies showed that all beta-fructans (including levan) are fermented by intestinal bacteria and can be classified as pro-biotics [bib_ref] The bifidogenic nature of chicory inulin and its hydrolysis products, Roberfroid [/bib_ref] , but levan has not been classified as a prebiotic fiber since in vivo studies on rats [bib_ref] In vitro digestibility and fermentability of levan and its hypocholesterolemic effects in..., Yamamoto [/bib_ref] did not support the proposition that it can be fermented by strains of bifidobacteria. We did not study the prebiotic characteristics of levan in colonic and stool cultures, but we did expect some impact of levan on gastrointestinal symptoms, which was not shown by our results. One possible explanation is that the usual Israeli diet has a relatively high fiber content as we saw in the pre-study diets of both groups. The findings of the current study showed a significant decrease in the blood pressure (within normal range) as result of orange juice consumption in normotensive subjects. Although it is a common belief that orange juice is able to lower blood pressure in hypertensive patients, we believe that this is the first controlled study to look into this subject. Based on the findings of our study, further research into the impact of orange juice on hypertension is warranted.
A very interesting question is the possible effect of sugar-induced juice on glucose control. Our study showed that despite a lower glycemic index in the pilot group, no influence of the juice on glucose level and HBA1C was demonstrated in either group. However, both groups included only healthy volunteers. Therefore the possible effect of sugar-induced juice on glucose control should be studies on diabetic patients or patients with border-line hyperglycemia.
The results of Yamamoto et al.'s study using a rat model suggested a hypocholesterolemic effect of levan [bib_ref] In vitro digestibility and fermentability of levan and its hypocholesterolemic effects in..., Yamamoto [/bib_ref]. The study of showed some positive effect of levan on weight, body fat, cholesterol profile and triglyceride level in Korean women [bib_ref] The effect of fructose polymer levan on the body fat accumulation and..., Kang [/bib_ref]. Our current study showed no similar effect of levan-supplemented or plain orange juice on the levels of cholesterol or triglycerides among healthy subjects with normal cholesterol and triglycerides levels. Further studies on the possible effects of orange juice alone and orange juice enriched with levan on hypertensive and hyperlipidemic patients might yield interesting findings. Also interesting is the possible effect of fiber on mineral absorption, as suggested by past animal studies [bib_ref] Effects of fructooligosaccharides on the absorption of iron, calcium and magnesium in..., Ohta [/bib_ref]. We found effects of levan on blood levels of any mineral (calcium, magnesium, iron, etc.). Finally, the sodium blood levels increased significantly in both our study and control groups (within normal limits). Since there were no differences between the groups, the effect must have been due to the orange juice itself.
# Conclusion
In summary, we found that daily consumption of a fiber-enriched, sugar-reduced orange juice for 8 weeks caused no significant impact on metabolic parameters and gastrointestinal symptoms in healthy participants compared to regular orange juice. Further studies on the effect of levan on obese, hypertensive and hyperlipidemic patients and on the possible effect of orange juice itself are warranted.
[table] Table 1: Comparison between regular juice and levan-enriched juice. [/table]
[table] Table 2: Demographics of the study population. [/table]
[table] Table 3: Results of mixed model for relative changes in gastrointestinal symptoms during 8 weeks of juice drinking. [/table]
[table] Table 4: Results of mixed model for relative changes in BMI, blood pressure and blood tests during 8 weeks of juice drinking. [/table]
[table] Table 5: Glycemic index of levan-enriched and regular juice. [/table]
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Modulating gene regulation function by chemically controlled transcription factor clustering
Supplementary Figure 1: Additional characterizations of rapamycin-controlled TF clustering. a, Representative confocal images of U2OS-7TetO cells under 0 nM or 100 nM rapamycin. Each cell image was overlaid with software-detected nucleus contour (blue) and detected TF clusters (red). Images were generated from maximum projection of confocal z-stack. b-c, Dot plots and boxplots showing quantifications of TF clustering. Detected numbers of TF clusters and the corresponding TF concentrations (quantified as mean nuclear EGFP intensities) in individual cells (with each dot representing one cell) are plotted for U2OS-7TetO cells under various indicated rapamycin concentrations (b). Mean cluster numbers are plotted inFig. 1e. Analogous plots for mean cluster diameter in individual cells are shown (c). Population-averaged cluster diameters are plotted inFig. 1f. The cell numbers are between 70 and 117 (see source data for the exact number). For boxplots, middle line indicates median and box ranges from first to third quartile, with whiskers extending up to 1.5 × interquartile range. d, Two-color construct for analyzing interactions between clustering mediator and the synthetic TF. Compared to the U2OS-7TetO system inFig. 1a, in this two-color construct each component was fused with distinct fluorescent proteins (top). Three representative cells were shown for 0 nM and 100 nM rapamycin conditions separately. Images were generated from maximum projection of confocal z-stack. From these results, it is evident that the formation of TF clusters is controlled by rapamycin-dependent mechanism. e, Schematic of the CHO-Gal4 system. The clustering mechanism is the same as the U2OS-7TetO system (i.e.,Fig. 1a). It should be noted that, in contrast to the U2OS-7TetO system where doxycycline is used for enabling the DNA binding capability of rTetR, here doxycycline is used to induce the expression of synthetic TF Gal4-VP64 and clustering mediator. f, Control experiments showing that rapamycin addition does not affect TF fluorescence (left) and the activation of downstream genes requires the DNA binding capability of the TF (right). g, FACS data gating strategy. Note that we only gated for singlets. Source data are provided. for examples of cells with images of separate colors. For each color, images were generated from maximum projection of confocal z-stack (Methods). Red arrow indicates overlapping TF clusters and DNA FISH signals. b, Histogram of the number of detected DNA FISH foci per cell. n = 16 cells. Mean number of dots per cell is 11.4. c, Histogram of the fraction of FISH foci co-localizing with TF clusters per cell. n = 16 cells. Mean overlap fraction is 14.7%. d, Boxplot comparing the intensity of TF clusters co-localizing versus non-colocalizing with DNA FISH foci in the prescence of doxycycline. The intensity of co-localized TF clusters or the mean intensity of the top 3 largest non-colocalizing TF clusters in the corresponding cell was used. 20 co-localized TF clusters from 16 cells were included in the plot. p value was calculated by paired two-sided t-test (t = -3.6846, df = 19). For boxplots, horizontal line indicates median and box ranges from first to third quartile, with whiskers extending up to 1.5 × interquartile range. Source data are provided. : Evidence supporting that TF clusters can activate the transcription of genome-integrated reporter genes. a-c, Additional characterizations of spatiotemporal interactions between TF clusters and nascent transcription sites. Time-lapse two-color snapshots of an example cell showing spatiotemporal interactions between TF clusters and four box-highlighted nascent transcription sites of reporter genes (a). Note that zoom-in views of the upper right corner of the cell were shown in . From two-color traces, the dwell time of TF clusters binding to reporter locus could be estimated (b, n = 32 events). Insert in b displays the histogram in log-linear scale to illustrate the exponential-like behavior. Schematics and example experimental traces of different modes of interaction between TF clusters and nascent transcription sites were shown in c. Each pair of experimental traces (GFP and mCherry) represents one reporter locus. Note that classification of interaction mode was purely empirical and may not represent the actual mode of interaction, because imaging was not continuous. See also Supplementary Movie 1. d-e, Quantifications of co-localization between TF cluster signals and nascent transcription signals. TF clusters were first identified in EGFP channel and their intensities were normalized by mean cellular EGFP intensity (Methods). The corresponding PCP intensities were calculated in mCherry channel and were normalized by mean cellular mCherry intensity. To control for by-chance co-localization, pseudo-TF clusters were generated as control (Methods). Data for the condition with doxycycline (0.3 μg/mL) are shown together with control data (d) or the condition without dox (e). Data points above threshold were identified as co-localization. The bootstrapped means of co-localization fractions are shown in . f-g, Analogous quantifications of co-localization by using the number of PCP foci as denominator (n = 615 cells). For boxplots, horizontal line indicates median and box ranges from first to third quartile, with whiskers extending up to 1.5 × interquartile range. h, For co-localized foci in the dox condition, PCP signal intensity and TF cluster intensity displayed an appearent negative correlation. Pearson correlation coefficient and p-value were extracted from corr function in Matlab (t = -3.685, df = 112) Source data are provided. a-e, Characterizations of the CHO-Gal4 system analogous to the U2OS-7TetO system, including gene regulation function (a), dissociation constant (b), Hill coefficient (c), bimodality index (d), and local sensitivity (e). Note that a gradient of doxycycline concentrations was used for generating a large range of TF expression levels for CHO-Gal4 system (Methods). Some of the plots in a were shown in [fig_ref] Supplementary, Figure 4: Characterization of transcriptional dynamics modulation by TF clustering [/fig_ref]. n = 2 biological replicates. f-i, Characterizations of the U2OS-1TetO system, including gene regulation function (f), dissociation constant (g), Hill coefficient (h), and bimodality index (i). Note that some of the plots in f were shown in [fig_ref] Supplementary, Figure 4: Characterization of transcriptional dynamics modulation by TF clustering [/fig_ref]. n = 2 biological replicates. Source data are provided. : A potential mechanism for the sustained transcriptional response conferred by TF clustering. a-b, Schematic for the potential mechanism underlying sustained transcriptional response conferred by TF clustering. As we have demonstrated, TF clustering results in an gene regulation function with decreased dissociation constant K d and increased ultrasensitivity (a). The different K d values lead to different thresholds of the TF level for the activation (and deactivation) of the target gene, resulting in a longer duration of activation at high TF clustering propensity (b). c-d, Simulations with experimental gene regulation functions recapitulated the sustained transcriptional activation. Two populations of cells (5000 cells each) with the same initial input (TF) level distribution at t =0 were simulated for the indicated time course. The production rates of the target protein were calculated by the experimentally measured gene regulation function at zero or 50 nM rapamycin . The half-life for both the TF and the target protein was estimated to be ~12 hours based on . Normalized population-averaged input (c) and output (d) signals are shown. e-f, Simulated outputs when assuming that TF clustering only modulated ultrasensitivity but not effective binding affinity (e) or that TF clustering only modulated effective binding affinity but not ultrasensitivity (f). Red lines are outputs from the assumed conditions.
[formula] 1.0 Target TF b c U2OS-7TetO U2OS-7TetO U2OS-7TetO U2OS-7TetO U2OS-7TetO U2OS-7TetO U2OS-7TetO U2OS-7TetO [/formula]
[fig] Figure 2: Evidence for doxycycline-dependent binding of TF clusters to reporter DNA loci. a, Three-color merged images of U2OS-7TetO cells under indicated conditions. Green: TF clusters. Purple: DNA fluorescence in situ hybridization (FISH) with probes targeting the reporter gene loci. Blue: DAPI staining of DNA. See [/fig]
[fig] Supplementary, Figure 4: Characterization of transcriptional dynamics modulation by TF clustering. a, Example trajectories showing the nascent transcriptional activities for all detected reporter's nascent sites inside single cells under indicated rapamycin concentrations. It is noted that the number of detected nascent sites increases from top to bottom. See also Supplementary Movie S2. b, Time to the first transcriptional burst post-doxycycline addition for each nascent site in cells under indicated rapamycin concentrations. Cell numbers are 36, 46, 40, and 37 from left to right. p values were calculated by one-way ANOVA and TukeyHSD. For boxplots, horizontal line indicates median and box ranges from first to third quartile, with whiskers extending up to 1.5 × interquartile range. Source data are provided, which contain details of statistical tests. [/fig]
[fig] Figure 5, Figure 6: Additional characterizations of the roles of TF clustering in the U2OS-7Te-tO system. a-b, Distributions of input (a) and output (b) signals under indicated rapamycin concentrations for the U2OS-7TetO system. The plots relate toFig. 4b. c, Bimodality indexes of the input and output distributions at indicated rapamycin concentrations. Green boxed region indicates 5 nM to 30 nM rapamycin conditions. Data are presented as mean ± S.D. (n = 3 biological replicates). d, Steady-state TF (EGFP) and reporter (iRFP) signals were quantified by flow cytometry at indicated rapamycin concentrations (with 0.3 μg/ml doxycycline) and were fitted by Hill function (red curve). Note that some of the plots were shown inFig. 4b. e-f, The modulation of dissociation constant (e) and ultrasensitivity (quantified by Hill coefficient, e) by TF clustering propensity. Both parameters were obtained from the fitted Hill functions. Green boxed regions indicate the same range as in panel c. Data are presented as mean ± S.D. (n = 3 biological replicates) g, Squared norm of the residual versus Hill coefficient for nonlinear curve fitting of the input-output data at indicated rapamycin concentrations. h, Quantification of local sensitivity in the dose resposne curve. Linear region (in log-log scale) of the dose response data of indicated rapamycin concentration was used for fitting. Data were binned before fitting to avoid sampling bias (Methods). i-j. Characteristics of the input and output distributions at indicated rapamycin concentrations, including standard deviation (i) and coefficient of variation (j). Data are presented as mean ± S.D. (n = 3 biological replicates) Source data are provided. Additional characterizations of the roles of TF clustering in two additional systems. [/fig]
[fig] Supplementary, Figure 7 Supplementary, Figure 8: Additional characterizations of sustained transcription responses conferred by TF clustering. a, Gated and ungated flow cytometry data from the experimental time course illustrated inFig. 5b. Note that gated and ungated iRFP distributions looked similar. b-c, Analogous plots toFig. 5c-f using ungated flow cytometry data. Data are presented as mean ± S.D. (n = 3 biological replicates). d, RT-qPCR quantification of the reporter gene using two primer sets at indicated time points during the above time course. Data are presented as mean ± S.D. (n = 3 biological replicates). e-h, Time-lapse imaging data further support sustained transcription responses conferred by TF clustering. CHO-Gal4 cells were subjected to a 12 hr pulse of doxycycline (0.1 μg/mL) treatment and fluorescence images of EGFP and iRFP were taken every hour. Processed data from indicated time points are shown (e). See Methods for details regarding image processing. Data were normalized in order to compare time scales. Results from three rapamycin conditions show that, while TF concentrations decayed similarly (f), the iRFP levels displayed more sustained activation for conditions with higher rapamycin concentrations (g). Each point indicates result from one field-of-view. Data are presented as mean ± S.D. n = 19, 20, 20 field-of-views for 0, 5, and 50 nM rapamycin, respectively. During the time course, the number of TF clusters per field-of-view decreased gradually (h, n = 8 filed-of-views). Source data are provided. Quantifications of the reporter genes' epigenetic states after transient signal by ChIP-qPCR. a-b, Experiment design. CHO-Gal4 cells were subjected to a 12 hr pulse of doxycycline (0.1 μg/mL) treatment and ChIP-qPCR quantifications were carried out at indicated time points (a) with four separate primer sets (b [/fig]
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## Phytoncides derived from pine tree
The pine tree is classified as any conifer in the genus Pinus of the family Pinaceae. Pinus is the sole genus in the subfamily Pinoideae. Pine includes conifers such as cedars, spruces, firs and pines. Of all the conifers, the pines have one of the largest distributions in the world, although they are found almost entirely in the northern hemisphere. They are found throughout much of the North America, China, South-East Asia including South Korea, Russia and Europe. Pines have been used in everyday life and throughout the world for their unique aroma and various therapeutic potentials. These are derived from pine essential oils, also known as phytoncide. Pine essential oils are mainly composed of monoterpene such as α-and β-pinene, 3-carene, limonene, and terpinene. These compounds have versatile properties such as anti-fungal, anti-inflammatory, anti-microbial, analgesic, and anti-stress.
α-Pinene [2,6,6,-trimethylbicyclo(3.1.1)-2-hept-2-ene)] is the most abundant terpene of pine essential oils and it has a hydrocarbon group of bicylic terpenes with a distinctive turpentine odor. It has been widely used as a food flavoring ingredient and component of perfumes. Besides its various biological properties including anti-microbial, hypertensive, anti-nociceptive, and anti-inflammatory, it also has an anxiolytic effect and sleep enhancing effect by inhalation and oral administration. 3-Carene (3,7,7-Trimethylbicyclo[4.1.0] hept-3-ene), the second abundant terpene of pine essential oils, is a bicyclic monoterpene consisting of fused cyclohexene and cyclopropane rings and has a sweet and pungent odor. It has been utilized as a raw material in perfumes, cosmetics, flavors and terpene resins, having various therapeutic properties like other pine essential oils. Recently, a sleep enhancing effect of 3-carene by oral administration just like α-pinene has been reported. Although pine essential oils have various biological and therapeutic effects, we focus on the sleep enhancing effects by the most abundant terpenes: α-pinene and 3-carene. Forest bathing reduced the cerebral activity and stress hormone Gomes-Carneiro et al.Salmonella/microsome assay Inhalation of α-pinene showed anxiolytic effect Detection of accumulated α-pinene in bran and liver Satou et al.Anxiety behavior (elevated plus maze) measure α-pinene detecting in organs by gas chromatography
Inhalation of α-pinene showed anxiolytic effect Detection of accumulated α-pinene in bran and liver Yamaoka et al.Sleep behavior measure Inhalation of α-pinene increased the REMS Yang et al.Sleep behavior measure Electrophysiology Molecular medeling
Oral administration of α-pinene increased the NREMS, prolonged the GABA response The binding site of α-pinene in GABA A BZD receptor was predicted Ocete et al.Anti-inflammatory assay Oral / intraperitoneally administration 3-carene showed the anti-inflammatory effect Woo et al.Electrophysiology
## Sleep behavioral study of phytoncide with eeg and emg
Sleep is divided into two broad stages: non-rapid eye movement sleep (non-REMS or NREMS) and rapid eye movement sleep (REMS). NREMS is further divided into 4 sub-stages of increasing depth leading to REMS. REMS represents a smaller portion of total sleep time, and is associated with desynchronized and fast brain waves, eye movements, loss of muscle tone, and suspension of homeostasis. Sleep stages can be monitored and measured by the well-known techniques; electroencephalogram (EEG) and electromyogram (EMG). EEG is an electrophysiological monitoring method to record electrical activity of the brain, measuring voltage fluctuations resulting from ionic current within the neurons of the brain. EEG can be utilized in various medical applications such as diagnosis of epilepsy, sleep disorders, depth of anesthesia, and brain death. In an EEG recording, the brain waves are a commonly used criteria for distinguishing sleep stages. Brain waves can be classified as alpha, beta, delta, and theta based on their own frequency and amplitude. EMG is an eletrodiagnostic medical technique for recording the electrical activity produced by skeletal muscles. EMG has various clinical and biomedical applications. It is used as a diagnostic tool for identifying neuromuscular diseases, motor disorders, and sleep stages.
Yamaoka et al.firstly reported the role of phytoncide from pine tree on sleep. They found that inhalation of (+)α-pinene in rats increased the duration of paradoxical sleep, also known as REMS through EEG and EMG recordings. In another study, an oral administration of (-)α-pinene showed a sedative effect at a low dose (< 25 mg/kg) and increased NREMS at high dose (100 mg/kg,in mice. The discrepancy of the effect of α-pinene on REMS and NREMS might be due to different administration methods, concentration, enantiomer type, or species. It has been previously established that sleep quantity, as measured by increased duration of NREMS, is enhanced by well-known hypnotics such as diazepam and zolpidem. However, a major pitfall of these hypnotics is that they reduce the delta activity, a high amplitude brain wave with a frequency of oscillation between 0.5 and 4 Hz in EEG recording. It has been widely accepted that delta activity is observed in the deep stage 3 of NREMS and is a critical index for the depth or intensity of NREMS. When we think about a good sleep or efficient sleep, we need to consider not only sleep quantity but also sleep quality. Although it is not trivial to define sleep quality in rodents, the delta activity would be a good indicator for the intensity of NREMS or sleep quality. Unlike conventional hypnotics such as zolpidem, α-pinene is shown to increase the duration of NREMS without affecting the delta activity. This is in great contrast to zolpidem, which causes increased duration of NREMS accompanied by a significantly reduced delta activity. These results suggest that α-pinene has a sleep-enhancing effect with an increase of sleep quantity, but no change in sleep quality. The sleep enhancing effect of α-pinene was fully blocked by flumazenil, an antagonist of the GABA A -BZD receptor, suggesting that α-pinene works as a modulator of GABA A -BZD receptor, just like diazepam and zolpidem. Based on the pharmacological evidence using flumazenil in EEG recording, the next obvious question is what is the molecular mechanism of α-pinene in action. This question can be addressed by the next scientific approach: electrophysiological recording from brain slices.
## Phytoncides as sleep aids
## Molecular mechanism of phytoncide through electrophysiological approach
It has been reported that hypnotics such as diazepam and zolpidem enhance GABAergic inhibitory signaling by prolonging the decay time constant of GABA A receptors in various brain region including thalamus, hippocampus, and neocortex. Recent studies also reported that phytoncides from pine tree such as α-pinene and 3-carene show the same prolonging effect on IPSCs (inhibitory postsynaptic currents,in CA1 hippocampusjust like diazepam and zolpidem. Interestingly, these two components of pine tree did not affect the amplitude and frequency of IPSCs, an indicator for changes in presynaptic and postsynaptic component such as the amount of presynaptic release of transmitters and the number of postsynaptic receptors in inhibitory signaling. The prolonging effect in inhibitory signaling by α-pinene and 3-carene was fully blocked by flumazenil, suggesting that these two phytoncide components modulate the GABA A BZD receptor with having the same molecular mechanism with diazepam and zolpidem.
Besides these monoterpenes from pine tree, other natural compounds from various plants such as borneol, verbenol, pinocar-veol, and isoliquiritigenin show the similar effect on GABAergic inhibitory signaling as a modulator for GABA A BZD receptor with sleep-enhancing effects. Based on these lines of evidence, we can make a generalization that if a certain drug or compound prolongs the decay time constant of IPSCs, we can predict that it could have a sleep-enhancing effect as a positive modulator of GABA A BZD receptor. This electrophysiological experiment could be a simple screening method for finding a potential hypnotic. In addition to the change in sIPSCs decay time, it has been reported the hyperpolarization of membrane potential from cortical neurons through the influx of chloride ion by hypnotics such as diazepam and zolpidem. Future experiments are needed to confirm the hyperpolarization of membrane potential in neurons by phytoncide including α-pinene and 3-carene. It has been reported the non-specific binding of hypnotics to other ion channels and transporters rather than GABA A receptor. Future experiments are required to test the non-specific binding of phytoncide relating to side effects such as cognitive defect.
## Structural biological approach through molecu-lar docking model
The sleep-enhancing effect of α-pinene and 3-carene has been demonstrated by pharmacological method using well-known modulator and antagonist of GABA A BZD receptor, sleep behavioral approach using EEG, and electrophysiological approach using patch-clamp technique. However, additional validation of the molecular mechanism with more advanced in-depth analysis is needed to demonstrate how and where the chemical constituent of each phytoncide bind to GABA A BZD receptor. This can be achieved by the latest tools available from the structural biology, the study of the molecular structure and dynamics of biological molecules, and how alterations in their structures affect their function.
It has been shown that α-pinene, zolpidem, and flumazenil bind to BZD binding site between α1 and 2-subunits in extracellular domain of GABA A receptor through the molecular modeling, encompassing theoretical and computational methods to model or mimic the behavior of the molecules. Contrary to zolpidem and flumazenil having π-π interaction with α1 and 2-subunits in extracellular domain of GABA A receptor, it was predicted that α-pinene makes strong hydrophobic interactions with aromatic residues of α1 and 2-subunits, suggesting the lower binding energy and affinity of α-pinene compared to zolpidem and flumazenil. Through the same approach, it was predicted that 3-carene also binds to BZD binding site between α1 and 2subunits in extracellular domain of GABA A receptor. 3-Carene has three different bonds including van der Waals, π-π, and π-σ interactions with α1 and 2-subunits. Based on Glide score (GScore), an empirical scoring function that approximates the ligand binding free energy, 3-carene (GScore: -5.39 kcal/ mol) has lower binding energy and affinity than α-pinene (GScore: -6.57 kcal/mol), zolpidem (GScore: -9.22 kcal/mol), and fluma- Phytoncides as Sleep Aids zenil (GScore: -8.78 kcal/mol). Through this molecular modeling, we can predict that α-pinene and 3-carene modulate the biologic function of GABA A receptor by directly binding at the BZD binding site.
It has been reported that α1/ α3 subunits of the GABA A receptor are predominant in the corticothalamic network, which is responsible for the generation of delta activity. α-Pinene and 3-carene have lower binding energy and affinity to α1 subunit of GABA A receptor through a hydrophobic interaction. This biomolecular property might be the reason that α-pinene did not affect delta activity indicating sleep quality. Future experiments are needed to identify the relationship between the binding property of hypnotics and the degree of side effects, including a decrease in sleep quality.
## Concluding remarks
We have discussed the sleep-enhancing effect of phytoncides from pine trees through behavioral, pharmacological, electrophysiological, and structural approaches. Through these approaches, it has been established that major phytoncides including α-pinene and 3-carene enhance the quantity of NREMS without affecting the sleep quality by prolonging GABAergic inhibitory signaling as a positive modulator of GABA A BZD receptor. Although other natural compounds from various plants can have a similar effect in sleep like phytoncide, we can obtain phytoncide' s beneficial effects including anxiolytic, anti-stress, and sleep enhancing effects from our daily life such as strolling in the woods or forest-bathing. It has been reported that conventional hypnotics such as diazepam and zolpidem have various side effects including cognitive impairment, tolerance, rebound insomnia upon discontinuation, abuse, and dependence liability. These side-effects are becoming increasingly serious as more people experience sleep disorders. Studies on the sleep-enhancing effects of α-pinene and 3-carene raise the possibility that these phytoncides might have less side-effects compared to the conventional hypnotics. Future work is needed to directly compare between phytoncides and existing hypnotics in terms of the side-effects. Furthermore, recent studies verify the effects of forest bathing scientifically and suggest screening tools for identifying prospective hypnotics from natural compounds. |
Methodological considerations of electron spin resonance spin trapping techniques for measuring reactive oxygen species generated from metal oxide nanomaterials
Qualitative and quantitative analyses of reactive oxygen species (ROS) generated on the surfaces of nanomaterials are important for understanding their toxicity and toxic mechanisms, which are in turn beneficial for manufacturing more biocompatible nanomaterials in many industrial fields. Electron spin resonance (ESR) is a useful tool for detecting ROS formation. However, using this technique without first considering the physicochemical properties of nanomaterials and proper conditions of the spin trapping agent (such as incubation time) may lead to misinterpretation of the resulting data. In this report, we suggest methodological considerations for ESR as pertains to magnetism, sample preparation and proper incubation time with spin trapping agents. Based on our results, each spin trapping agent should be given the proper incubation time. For nanomaterials having magnetic properties, it is useful to remove these nanomaterials via centrifugation after reacting with spin trapping agents. Sonication for the purpose of sample dispersion and sample light exposure should be controlled during ESR in order to enhance the obtained ROS signal. This report will allow researchers to better design ESR spin trapping applications involving nanomaterials.To further promote industries that rely on nanotechnology, it is important to understand not only the characteristics and potentials of such materials, but also their toxicity. The field of toxicology has produced a wealth of information relating to the toxicity or toxic mechanisms of nanomaterials, which have expanded and refined the industrial uses of such materials. In recent decades, these efforts have been highly beneficial to understanding the toxicities and toxic mechanisms of various nanomaterials and to safely and responsibly develop their related technologies. Nevertheless, nanomaterials have many different forms and applications, and this diversity must be considered when studying their toxicity. In particular, in the initial and current state of nanotoxicology, various nanomaterial properties such as size, shape, and charge must be considered due to their pivotal roles in influencing material toxicity. Therefore, determining the physicochemical properties of nanomaterials is critical in evaluating their overall toxicity. In the relevant biological and physicochemical analyses, it is essential to not only produce relevant data but to understand the limitations of existing methods.Reactive oxygen species (ROS) are highly reactive substances that are potentially toxic to living organisms. They can cause detrimental effects in cells through oxidative damage of biomolecules (proteins, lipids, and nucleic acids) or through disruption of cell signaling pathways 1 . ROS is considered one of the most important sources
Qualitative and quantitative analyses of reactive oxygen species (ROS) generated on the surfaces of nanomaterials are important for understanding their toxicity and toxic mechanisms, which are in turn beneficial for manufacturing more biocompatible nanomaterials in many industrial fields. Electron spin resonance (ESR) is a useful tool for detecting ROS formation. However, using this technique without first considering the physicochemical properties of nanomaterials and proper conditions of the spin trapping agent (such as incubation time) may lead to misinterpretation of the resulting data. In this report, we suggest methodological considerations for ESR as pertains to magnetism, sample preparation and proper incubation time with spin trapping agents. Based on our results, each spin trapping agent should be given the proper incubation time. For nanomaterials having magnetic properties, it is useful to remove these nanomaterials via centrifugation after reacting with spin trapping agents. Sonication for the purpose of sample dispersion and sample light exposure should be controlled during ESR in order to enhance the obtained ROS signal. This report will allow researchers to better design ESR spin trapping applications involving nanomaterials.
To further promote industries that rely on nanotechnology, it is important to understand not only the characteristics and potentials of such materials, but also their toxicity. The field of toxicology has produced a wealth of information relating to the toxicity or toxic mechanisms of nanomaterials, which have expanded and refined the industrial uses of such materials. In recent decades, these efforts have been highly beneficial to understanding the toxicities and toxic mechanisms of various nanomaterials and to safely and responsibly develop their related technologies. Nevertheless, nanomaterials have many different forms and applications, and this diversity must be considered when studying their toxicity. In particular, in the initial and current state of nanotoxicology, various nanomaterial properties such as size, shape, and charge must be considered due to their pivotal roles in influencing material toxicity. Therefore, determining the physicochemical properties of nanomaterials is critical in evaluating their overall toxicity. In the relevant biological and physicochemical analyses, it is essential to not only produce relevant data but to understand the limitations of existing methods.
Reactive oxygen species (ROS) are highly reactive substances that are potentially toxic to living organisms. They can cause detrimental effects in cells through oxidative damage of biomolecules (proteins, lipids, and nucleic acids) or through disruption of cell signaling pathways 1 . ROS is considered one of the most important sources of toxic mechanisms in various nanomaterials. Many reports suggest that the entrance of nanomaterials into the cell may lead to the formation of ROS. In addition, the formation of ROS in the cell can induce toxicity through a number of complicated pathways [bib_ref] Nickel oxide nanoparticles exert cytotoxicity via oxidative stress and induce apoptotic response..., Ahamed [/bib_ref] [bib_ref] Cytotoxicity, oxidative stress, apoptosis and the autophagic effects of silver nanoparticles in..., Lee [/bib_ref] [bib_ref] In vitro cytotoxicity and genotoxicity studies of titanium dioxide (TiO 2 )..., Hamzeh [/bib_ref]. Therefore, it is important to measure and quantify ROS formation in order to understand ROS-related toxicity in nanomaterials 5 . ROS typically have a short lifespan, making their direct detection and determination very difficult and often impossible [bib_ref] Getting a better estimate of an atmospheric radical, Isaksen [/bib_ref] [bib_ref] Mechanistic characterization of titanium dioxide nanoparticle-induced toxicity using electron spin resonance, Li [/bib_ref]. The use of fluorescent probes, such as dichlorodihydrofluorescein and hydroethidine, as well as dihydrorhodamine and chemiluminescent methods, provides simple ways of detecting free radicals and ROS in cellular systems. Nevertheless, these methods have limitations and many sources of artifacts [bib_ref] Fluorescent and luminescent probes for detection of reactive oxygen and nitrogen species, Chen [/bib_ref] [bib_ref] Quality control of reactive oxygen species measurement by luminol-dependent chemiluminescence assay, Kobayashi [/bib_ref] [bib_ref] Photogenerated charge carriers and reactive oxygen species in ZnO/Au hybrid nanostructures with..., He [/bib_ref].
Electron spin resonance (ESR) (also called electron paramagnetic resonance or EPR) is a powerful technique for studying chemical species that have one or more unpaired electrons. ESR spectroscopy has become a direct and potent (robust-Rev1) method for detecting free radicals that are chemically generated or formed in biological systems, and in nanotoxicology this technique has been employed for detecting ROS [bib_ref] Phototoxicity of nano titanium dioxides in HaCaT keratinocytes-generation of reactive oxygen species..., Yin [/bib_ref] [bib_ref] Enhanced inactivation of bacteria by metal-oxide nanoparticles combined with visible light irradiation, Lipovsky [/bib_ref] [bib_ref] The effect of tungstate nanoparticles on reactive oxygen species and cytotoxicity in..., Dunnick [/bib_ref]. ESR techniques with various spin trapping agents are used to detect specific free radicals and to produce a relatively stable and distinct spin adduct that can be identified and quantified by ESR [bib_ref] Phototoxicity of nano titanium dioxides in HaCaT keratinocytes-generation of reactive oxygen species..., Yin [/bib_ref]. Although ESR with spin trapping agents is a very robust and valuable method of detecting ROS, selection of the correct specific type of spin trapping agent, sample preparation method, and incubation time between spin trapping agents and target materials must be addressed in greater detail. He et al., for example, contended that light exposure may affect the formation of ROS in metal nanoparticles, and that it also influences the formation of free radicals [bib_ref] Photogenerated charge carriers and reactive oxygen species in ZnO/Au hybrid nanostructures with..., He [/bib_ref] [bib_ref] Electron spin resonance spectroscopy for the study of nanomaterial-mediated generation of reactive..., He [/bib_ref]. Moreover, ultrasound was reported by Makino, Mossoba and Riesz to affect overall sample preparation [bib_ref] Chemical effects of ultrasound on aqueous solutions. Evidence for hydroxyl and hydrogen..., Makino [/bib_ref]. In this study, we suggest methodological considerations to follow in regards to the magnetism of nanomaterials, incubation time of spin trapping agents, and various other factors that can lead to distortion of ESR signals of nanomaterials such as light exposure and sonication, which are often used carelessly in ESR for measuring the ROS generated from metal oxide nanomaterials.
# Results
Spin adduct stability as a function of incubation time. We analyzed the stability of the spin adduct as a function of the incubation time in a positive control system [fig_ref] Figure 1: Stability reviews of each spin adduct according to incubation time in each... [/fig_ref]. In the case of the DMPO-OH adduct, the signal intensity increases until approximately 150 min in the Fenton reaction system. From 150 min to approximately 4 h, the signal intensity is saturated [fig_ref] Figure 1: Stability reviews of each spin adduct according to incubation time in each... [/fig_ref]. In the case of the BMPO-OOH adduct, the signal intensity increases until approximately 12 min, and then slowly decreases over time in the hypoxanthine-xanthine oxidase system [fig_ref] Figure 1: Stability reviews of each spin adduct according to incubation time in each... [/fig_ref]. However, the TPC-1 O 2 adduct is very stable until approximately 4 h [fig_ref] Figure 1: Stability reviews of each spin adduct according to incubation time in each... [/fig_ref]. Based on our results, determination of the incubation time between the spin trapping agent and test materials may be important in future quantitative analysis. These results allow us to determine an optimal reaction time for spin trapping reactions, respectively.
## Tem characterization of test nanomaterials. transmission electron microscopy (tem) analysis
was performed to confirm the particle morphology and size of the nanomaterials. As shown in [fig_ref] Figure 2: TEM images of metal oxide nanomaterials [/fig_ref] Magnetic nanomaterials and centrifugation. Nanomaterials have many physicochemical properties; among them, magnetism can impact the ESR signal of the material. We compared the ESR signal before and after removing nanomaterials by centrifugation or filtration in the case of CoFe 2 O 4 (a magnetic nanomaterial) in a positive control system. Because ESR signals of CoFe 2 O 4 samples were not detected based on hydroxyl radical signals (DMPO-OH adducts) after mixing the nanomaterials and spin trapping agent (DMPO) for 3 h (data not shown), samples were detected using a positive control system. ESR signals were distorted in the case of well-dispersed CoFe 2 O 4 [fig_ref] Figure 3: ESR spectra confirming the removal effect of nanomaterials on sample preparation [/fig_ref]. This ESR signal was tilted to one side because of contributions from both the spin adduct spectrum and a small portion (335 ± 10 mT) of the very broad (few hundred mT) intrinsic ESR spectrum of the unpaired electrons from CoFe 2 O 4 . No ESR signal was present for DMPO-OH adducts (Quartet with 1:2:2:1 signal intensity) despite the positive control system. However, after removing the well-dispersed CoFe 2 O 4 by centrifugation, the ESR signal was not tilted and DMPO-OH adducts were detected. and SiO 2 nanomaterials were detected in positive control system and ZnO nanomaterials were detected without positive control system. The nanomaterials were removed by centrifugation (at 12700 × g for 10 min) or filtration (with 0.2 μ m pore size).
In the case of sedimented CoFe 2 O 4 , there is no distortion of the ESR signals [fig_ref] Figure 3: ESR spectra confirming the removal effect of nanomaterials on sample preparation [/fig_ref]. Based on this result, we conclude that well-dispersed nanomaterials with magnetic properties should not be directly applied in ESR due to interference issues [fig_ref] Figure 3: ESR spectra confirming the removal effect of nanomaterials on sample preparation [/fig_ref].
To evaluate the interference effects of non-magnetic nanomaterials (ZnO and SiO 2 ), ESR spectra were collected before and after removing the nanomaterials by centrifugation or filtration. Here, there is no difference in ESR signal for samples with and without non-magnetic nanomaterials [fig_ref] Figure 3: ESR spectra confirming the removal effect of nanomaterials on sample preparation [/fig_ref]. Based on our results, there is no adverse effect of removing nanomaterials after incubating the spin trapping agent; therefore we suggest that removal of well-dispersed nanomaterials having magnetic properties by centrifugation could be applied to ESR analysis.
Effects of light exposure during sample preparation on ESR spectra. Each nanomaterial was exposed to various light sources, such as UV, natural light, visible light, and dark conditions in the presence of a spin trapping agent (DMPO). The signal of the associated hydroxyl radical adduct was then recorded to determine if any changes occurred. As shown in [fig_ref] Figure 4: ESR spectra confirming the effects of light exposure interference on sample preparation... [/fig_ref] , all ESR spectra increase in intensity under UV (280-360 nm) conditions as compared to dark conditions. The ESR spectrum of distilled water (DW) without nanomaterials increases under UV conditions as well. Excited H 2 O* generates hydrogen atoms (H - ) and hydroxyl radicals ( - OH) [bib_ref] Generation mechanism of hydroxyl radical species and its lifetime prediction during the..., Attri [/bib_ref] , and these hydroxyl radicals are subsequently trapped by DMPO. For this reason, the ESR spectrum of the DMPO-OH adducts increases in intensity.
In the case of TiO 2 , the ESR spectrum increases slightly in visible light (2.03 × 10 13 spins) and in natural light (2.00 × 10 13 spins). For ZnO, the ESR spectrum increases in order of dark conditions (2.82 × 10 13 spins) < visible light (3.37 × 10 13 spins) < natural light (9.43 × 10 13 spins) < UV (1.84 × 10 14 spins), and is more strongly sensitized to light compared to other nanomaterials. The ESR spectra of ZnO and TiO 2 with photocatalytic behavior increase remarkably under UV conditions. Based on our results, exposure to specific light sources can affect ESR spectra not only for nanomaterials, but for DW as well.
Effects of ultrasound sonication during sample preparation on ESR spectra. Sonication is commonly used in nanotechnology to evenly disperse nanomaterials in liquid media. As shown in [fig_ref] Figure 5: ESR spectra confirming interference effects of ultrasonic dispersion on sample preparation with... [/fig_ref] , ESR spectra were newly generated after ultrasonic dispersion for D.W., solutions of Al 2 O 3 , TiO 2 , and SiO 2 . In the case of ZnO, the ESR spectrum increases 4.8× when prepared by ultrasonic dispersion compared to a solution without sonication (1.36 × 10 14 spins vs. 2.82 × 10 13 spins). Therefore, because ROS signals can be generated by ultrasonic dispersion, such dispersion should not be used for detecting ROS generated from the nanomaterial itself.
# Discussion
ROS are a primary source of nanomaterial toxicity that can form after the exposure of nanomaterials to various elements, and are inherent to the surface conditions of the nanomaterials. However, the in situ identification and quantification of ROS in nanomaterials has some obstacles, such as their interactions with repair systems and other unknown factors. To avoid these problems, ROS must be analyzed directly during their formation from nanomaterials. In addition, the direct identification and quantification of ROS by ESR is important in understanding the toxic mechanisms of nanomaterials and in product design integrating these nanomaterials. In particular, the various factors that can affect ESR signals must be considered, such as the characteristics of the parent nanomaterial, spin trapping agent incubation time, and sample preparation. In this report, we examine the magnetic properties, photo catalytic activity, and dispersion method of such nanomaterials and how they can impact ESR analysis, as well as examining samples with and without the parent nanomaterials (removed by centrifugation or filtration) [fig_ref] Figure 3: ESR spectra confirming the removal effect of nanomaterials on sample preparation [/fig_ref].
Each spin trapping agents should be applied with a proper incubation time when using ESR to quantify ROS from nanomaterials. In this case, the life span of each spin trapping agent should be considered. Based on our results, the ESR signal intensity of the BMPO adduct decreases after the peak value, in contrast with the DMPO and TPC adducts [fig_ref] Figure 1: Stability reviews of each spin adduct according to incubation time in each... [/fig_ref]. In our results, spin trapping agents have different signal intensities depending on their incubation time [fig_ref] Figure 1: Stability reviews of each spin adduct according to incubation time in each... [/fig_ref] , emphasizing the importance of this parameter.
In the ESR analysis of various metal oxide nanomaterials, well-dispersed nanomaterials such as CoFe 2 O 4 should not be used for ESR directly due to their inherent magnetic properties, which can affect the ESR data. However, removing the nanomaterials after the reaction of the spin trapping agent is very effective in these cases [fig_ref] Figure 3: ESR spectra confirming the removal effect of nanomaterials on sample preparation [/fig_ref]. The photocatalytic activity of the nanomaterials should be additionally considered for ESR. In our results and various reports, the ESR signal intensity of nanomaterials with photocatalytic activity differs before and after light exposure [fig_ref] Figure 4: ESR spectra confirming the effects of light exposure interference on sample preparation... [/fig_ref] [bib_ref] Photogenerated charge carriers and reactive oxygen species in ZnO/Au hybrid nanostructures with..., He [/bib_ref] [bib_ref] Electron spin resonance spectroscopy for the study of nanomaterial-mediated generation of reactive..., He [/bib_ref]. Based on this result, exposure to various light sources should be also controlled when collecting nanomaterial ESR signals. During sample preparation, ultrasonication can also enhance ESR signals [fig_ref] Figure 5: ESR spectra confirming interference effects of ultrasonic dispersion on sample preparation with... [/fig_ref]. Therefore, ultrasonic processes should be carefully applied to ESR, especially in quantitative studies. In our well-documented experimental conditions, only the hydroxyl radical was observed from ZnO nanomaterials. In other cases, there was no observed ROS from nanomaterials [fig_ref] Figure 6: ESR spectra in accordance with well-documented methods in the sample preparation of... [/fig_ref].
# Conclusions
The qualitative and quantitative analyses of ROS generated on nanomaterial surfaces are important for understanding and predicting their toxicity and toxic mechanisms. To this end, ESR spin trapping techniques are very useful tools for detecting ROS formation. However, applying these without considering the physicochemical properties of the nanomaterials and the sampling conditions can lead to data misinterpretation. This study enables researchers to design accurate applications of ESR spin trapping techniques with nanomaterials and to interpret the results of toxicity studies [fig_ref] Figure 7: Methodological considerations for using ESR spin-trapping techniques with nanomaterials [/fig_ref].
# Materials and methods
Materials and instrumental preparation. The reaction mixture contained 10 mM TPC and 0.1 mM Rose Bengal in DW; it was illuminated with continuous light (λ max = approximately 550 nm) using an LED lamp for 10 min. ESR signals were collected every 3 min for 1 h.
Size and morphology measurements of nanomaterials. The morphology and nanostructure of CoFe 2 O 4 (well-dispersed and sedimented), ZnO, Al 2 O 3 , TiO 2 , and SiO 2 were characterized by energy-filtering transmission electron microscopy (EF-TEM) using a LIBLA 120 microscope (Carl Zeiss, Oberkochem, Germany) at an accelerating voltage of 120 kV. The suspension (4 mg/mL) of nanomaterials was prepared in methanol and sonicated for 30 s. A portion of the nanomaterial suspension was deposited on a 300-mesh carbon-coated copper grid and dried at room temperature overnight before examination.
## Esr measurements of magnetic nanomaterials.
To estimate the effect of magnetic properties on the nanomaterials, we prepared two different states of CoFe 2 O 4 , those being well-dispersed and sedimented materials. Samples were examined by ESR after mixing with a spin trapping agent (DMPO) for 3 h, at which point the hydroxyl radical (- OH) signal was not detected (data not shown). For this reason, samples were detected using a positive control system. After ESR analysis of both samples, we removed the CoFe 2 O 4 by centrifugation (12,700 × g for 10 min), and then re-analyzed with ESR. Then, to evaluate the interference effects of non-magnetic nanomaterials (ZnO and SiO 2 ), ESR spectra were analyzed before and after removing the nanomaterials by centrifugation or filtration. As mentioned above, SiO 2 was examined with a positive control system, while ZnO nanomaterials were detected without this positive control system. ESR measurement to identify light source interference. Each nanomaterial suspension with a spin trapping agent (DMPO) was exposed to UVB (λ max = 280-360 nm), natural light, visible light (λ max = 670 nm), and dark conditions for 3 h, respectively. The signal of hydroxyl radical adduct was recorded to observe and changes compared to dark conditions. ESR measurements to identify interference by using ultrasonic dispersion. Mixtures with nanomaterials and spin trapping agent were immersed in an ultrasonic bath (42 kHz, 135 W) for 10 min after reaction for 3 h. All steps were performed in dark conditions. For each mixture, the signal of the hydroxyl radical adduct was recorded to verify if the ultrasonic bath had any effects compared to the unapplied mixture.
## Quantification of ros.
The radical concentrations in the samples were determined by comparing the area of the absorption peak with that of a standard sample (TEMPOL), with 3.0 × 10 14 spins in 1.0 × 10 −3 mM TEMPOL as a reference point. Spin counts were calculated from the area of the absorption peak of the Mn 2+ marker and the signal by double integration of the ESR spectrum. The radical number of the test sample could be calculated relative the area of the standard sample [bib_ref] The free radical species in polyacrylonitrile fibers induced by g-radiation and their..., Weihua [/bib_ref] [bib_ref] The application of ESR spin-trapping technique to the evaluation of SOD-like activity..., Keiichi [/bib_ref] [bib_ref] Kinetics and mechanisms of the sonolytic destruction of non-volatile organic compounds: investigation..., Nam [/bib_ref]. In summary, the standard sample (St) and test sample (T) should be measured with the same ESR spectrometer parameters, respectively. I Double integrating the spectra over the same scan range allows for the quantification of the test sample as
[fig] Figure 1: Stability reviews of each spin adduct according to incubation time in each positive control system. (a) Stability review of the DMPO-OH adduct generated by the Fenton reaction in the presence of DMPO, (b) Stability review of the BMPO-OOH adduct by the hypoxanthine-xanthine oxidase system in the presence of BMPO, (c) Stability review of the TPC-1 O 2 adduct generated by the Rose Bengal photosensitizer in the presence of TPC. Scientific RepoRts | 6:26347 | DOI: 10.1038/srep26347 [/fig]
[fig] Figure 2: TEM images of metal oxide nanomaterials. (a) Well-dispersed CoFe 2 O 4 (20.4 ± 10.3 nm), (b) Sedimented CoFe 2 O 4 (48.3 ± 9.7 nm), (c) ZnO nanorods (width: 16.2 ± 1.3 nm, length: 50.8 ± 11.8 nm), (d) Al 2 O 3 (15.6 ± 5.4 nm), (e) TiO 2 (26.5 ± 6.7 nm), (f) SiO 2 (72.9 ± 4.0 nm). Scientific RepoRts | 6:26347 | DOI: 10.1038/srep26347 [/fig]
[fig] Figure 3: ESR spectra confirming the removal effect of nanomaterials on sample preparation. (a) Welldispersed magnetic nanomaterials (CoFe 2 O 4 ), (b) Sedimented magnetic nanomaterials (CoFe 2 O 4 ). (c) Welldispersed non-magnetic nanomaterials (SiO 2 ), (d) Sedimented non-magnetic nanomaterials (ZnO). CoFe 2 O 4 [/fig]
[fig] Figure 4: ESR spectra confirming the effects of light exposure interference on sample preparation with DMPO (UV, natural light, visible light, and dark conditions from above). (a) ESR spectrum of negative control (DW), (b) ESR spectrum of ZnO, (c) ESR spectrum of Al 2 O 3 , (d) ESR spectrum of TiO 2 , (e) ESR spectrum of SiO 2 . Scientific RepoRts | 6:26347 | DOI: 10.1038/srep26347 [/fig]
[fig] Figure 5: ESR spectra confirming interference effects of ultrasonic dispersion on sample preparation with DMPO (upper: with sonication; bottom: without sonication). (a) ESR spectrum of negative control (DW), (b) ESR spectrum of ZnO, (c) ESR spectrum of Al 2 O 3 , (d) ESR spectrum of TiO 2 , (e) ESR spectrum of SiO 2 . trapping agents were 5,5-dimethyl-1-pyrroline N-oxide (DMPO) for • OH, 5-tert-butoxycarbonyl-5-methyl-1-pyrroline-N-oxide (BMPO) O 2 − , and 2,2,5,5-tetramethyl-3-pyrroline-3-carboxamide (TPC) for 1 O 2 . The nitrone spin-trap reagents 5,5-dimethyl-1-pyrroline N-oxide (DMPO, 99.9%) and 5-tert-butoxycarbonyl-5 -methyl-1-pyrroline-N-oxide (BMPO) were purchased from Dojindo (Tokyo, Japan). Sedimented CoFe 2 O 4 , 2,2,5,5-tetramethyl-3-pyrroline-3-carboxamide (TPC), iron(II) sulfate (FeSO 4 ), hydrogen peroxide (H 2 O 2 ), hypoxanthine (HPX), xanthine oxidase (XOD), diethylenetriaminepentaacetic acid (DTPA), phosphate buffer solution, dimethyl sulfoxide (DMSO) and Rose Bengal were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). 4-hydroxyl-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL) was purchased from JEOL (Tokyo, Japan). Syringe filters (0.20 μ m pore size) were purchased from Sartorius (Goettingen, Germany). An ultraviolet (UV)-B lamp (1.6 W, 0.170 A, G8T5E) was purchased from Sankyo Denki (Tokyo, Japan). The visible-light lamp (6 W, 60 mA, SSL-012458) and green lamp (6 W, 60 mA, ST-5030) were purchased from STECH LED (Gyeonggi-do, Korea).ESR measurements. The ESR signals of samples were acquired using a JES-TE200 ESR spectrometer (JEOL, Tokyo, Japan) with an X-band standard frequency of 8.8-9.6 GHz. To identify the peaks, the signal components were analyzed by the ES-IPRITS data system with version 3.00 analysis software installed in the ESR instrument. The following ESR parameters were used: a frequency of 9.42 GHz, center field of 335 ± 10 mT, modulation frequency of 100 kHz, time constant of 0.03 s, and power of 5.00 mW. Aqueous samples were loaded into a LC-12 aqueous quartz flat cell (JEOL)17 .This reaction mixture contained 100 mM phosphate buffer (pH 7.4) with 25 μ M DTPA, 0.125 mM hypoxanthine, 50 mM BMPO and 0.125 units/mL of xanthine oxidase. The reaction began with the addition of xanthine oxidase, and ESR signals were collected every 10 min for 4 h.Singlet oxygen ( 1 O 2 ). The singlet oxygen was generated from the Rose Bengal photosensitizer. When photoexcited, this photosensitizer transfers its energy to the oxygen-producing singlet oxygen (reactions 3 and 4)20 . [/fig]
[fig] Figure 6: ESR spectra in accordance with well-documented methods in the sample preparation of nanomaterials (negative control, ZnO, Al 2 O 3 , TiO 2 , and SiO 2 from above). (a) ESR data of hydroxyl radical (•OH) production in the presence of DMPO with each nanomaterial, (b) ESR data on superoxide anion radical (O 2 − ) production in the presence of BMPO with each nanomaterial, (c) ESR data on singlet oxygen ( 1 O 2 ) production in the presence of TPC with each nanomaterial. [/fig]
[fig] Figure 7: Methodological considerations for using ESR spin-trapping techniques with nanomaterials.Scientific RepoRts | 6:26347 | DOI: 10.1038/srep26347 [/fig]
[fig] A: St : The area of absorption peak with a standard sample (1.0 × 10 −3 mM TEMPOL) T : The area of absorption peak with a test sample M St : The area of absorption peak of Mn 2+ marker with a standard sample M T : The area of absorption peak of Mn 2+ marker with a test sample N T : The radical number of test sample. [/fig]
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Does the bracket–ligature combination affect the amount of orthodontic space closure over three months? A randomized controlled trial
Objective: To investigate the effect of bracket-ligature combination on the amount of orthodontic space closure over three months.Design: Randomized clinical trial with three parallel groups.Setting: A hospital orthodontic department (Chesterfield Royal Hospital, UK).Participants: Forty-five patients requiring upper first premolar extractions.Methods: Informed consent was obtained and participants were randomly allocated into one of three groups: (1) conventional pre-adjusted edgewise brackets and elastomeric ligatures; (2) conventional pre-adjusted edgewise brackets and Super SlickH low friction elastomeric ligatures; (3) Damon 3MXH passive self-ligating brackets. Space closure was undertaken on 0.01960.025-inch stainless steel archwires with nickel-titanium coil springs. Participants were recalled at four weekly intervals. Upper alginate impressions were taken at each visit (maximum three). The primary outcome measure was the mean amount of space closure in a 3-month period.Results: A one-way ANOVA was undertaken [dependent variable: mean space closure (mm); independent variable: group allocation]. The amount of space closure was very similar between the three groups (1 mm per 28 days); however, there was a wide variation in the rate of space closure between individuals. The differences in the amount of space closure over three months between the three groups was very small and non-significant (P50.718).Conclusion:The hypothesis that reducing friction by modifying the bracket/ligature interface increases the rate of space closure was not supported. The major determinant of orthodontic tooth movement is probably the individual patient response.
# Introduction
Proponents of self-ligating brackets suggest that low levels of friction between the archwire and bracket might increase the rate of tooth movement and hence reduce orthodontic treatment times compared with conventional brackets systems. [bib_ref] Treatment time, outcome, and patient satisfaction comparisons of Damon and conventional brackets, Eberting [/bib_ref] [bib_ref] Self-ligating brackets and treatment efficiency, Harradine [/bib_ref] One stage that might lead to prolonged treatment with fixed appliances is the closure of residual spaces following dental extraction to alleviate crowding or to reduce an increased overjet/overbite. Space closure can be undertaken with looped archwires; however, the introduction of the pre-adjusted edgewise appliance allows the use of sliding mechanics, which is simpler for the patient to maintain, has less potential for gingival trauma and is easier for the orthodontist to institute when compared to the bending of loops. The drawback of sliding mechanics is that it can be hindered by resistance arising from friction, binding, and notching that may contribute to slow tooth movement and prolonged treatment times.
In addition to self-ligating brackets various other products have been developed that claim to reduce static and/or dynamic friction between the bracket/archwire/ ligature interfaces and thereby theoretically enable more rapid tooth movements. [bib_ref] Physical properties of conventional and Super Slick elastomeric ligatures after intraoral use, Crawford [/bib_ref] [bib_ref] Clinical efficiency of nonconventional elastomeric ligatures in the canine retraction phase of..., Dholakia [/bib_ref] Super SlickH elastomeric ligatures have a covalently bonded Metafix coating to reduce friction, but laboratory tests have provided conflicting results. [bib_ref] Evaluation of methods of archwire ligation on frictional resistance, Khambay [/bib_ref] [bib_ref] Resistance to sliding with 3 types of elastomeric modules, Griffiths [/bib_ref] [bib_ref] A comparison of different ligation methods on friction, Hain [/bib_ref] In contrast, laboratory tests have consistently shown that the friction between self-ligating brackets and archwires is greatly reduced; however, there is currently limited evidence that this is translated into more rapid tooth movement in the clinical environment. [bib_ref] Systematic review of self-ligating brackets, Chen [/bib_ref] [bib_ref] Self-ligating brackets in orthodontics. A systematic review, Fleming [/bib_ref] The aim of this clinical study was to investigate the amount of active orthodontic space closure over 3 months with different bracket/ligature combinations. The null hypothesis was that there was no difference in the rate of space closure in patients treated with fixed orthodontic appliances using conventional pre-adjusted edgewise brackets and elastomeric ligatures compared with patients treated with conventional pre-adjusted edgewise brackets and Super SlickH elastomeric ligatures or Damon 3MXH self-ligating brackets.
# Participants and methods
The study was a single-blinded, randomized controlled clinical trial of parallel group design. Potential patients were screened at their first appointment in the Orthodontic Department and those that fulfilled the inclusion criteria were provided with verbal and written information about the study. They were given at least 1 week to consider whether or not to participate. At a subsequent review appointment, written consent was obtained from those who agreed to take part and they were randomly allocated to one of three groups. Randomization was carried out by one researcher not involved in recruiting patients to the study (PEB) using computer-generated random numbers. To ensure equal numbers, randomization was undertaken in three blocks of 3 (one of each group in a random sequence) and 6 (two of each group in a random sequence) and two blocks of 9 (three of each group in a random sequence). The blocks were placed in a random order. The allocations were concealed in consecutively numbered, sealed opaque envelopes, which were not opened until the patient had been enrolled into the study and consent obtained.
Group 1: Conventional brackets and elastomeric ligatures: Patients allocated to this group received conventional pre-adjusted edgewise brackets (American Orthodontics, Bucks, UK; 0.02260.028-inch; MBT prescription) bonded in both arches and the archwires were retained with conventional elastomeric ligation (American Orthodontics).
Group 2: Conventional brackets and Super SlickH elastomeric ligatures: Patients allocated to this group received conventional pre-adjusted edgewise brackets (American Orthodontics; 0.02260.028-inch; MBT prescription) bonded in both arches and the archwires were retained with Super SlickH elastomeric ligatures (TP Orthodontics, La Porte, IN, USA).
Group 3: Self-ligating brackets: Patients allocated to this group received Damon 3MXH passive self-ligating brackets (Ormco Corporation, Orange, CA, USA) bonded in both arches.
In all patients, bonded buccal tubes were used instead of molar bands, as it allowed simpler identification of the buccal groove of the first molar. Following extraction of maxillary first premolars, Group 1 and 2 underwent initial alignment with 0.016-inch and 0.01860.025-inch nickel-titanium archwires prior to placement of a 0.01960.025-inch rectangular stainless steel archwire. Group 3 received a series of copper nickel-titanium -0.014-inch, 0.01460.025-inch, 0.01860.025-inch Damon archwires (Ormco, Europe) for initial alignment according to the manufacturer's recommendation, followed by a 0.01960.025-inch stainless steel working archwire. It was not possible to mask the operator to group allocation.
Prior to space closure, in all participants the 0.01960.025-inch stainless-steel wires was in situ and passive for at least 6 weeks and the overbite reduced to normal (2-3 mm). Space closure involved en masse movement of the incisors and canines against the premolars and molars using 6 mm nickel-titanium coil springs (American Orthodontics). These were placed on the buccal hook of the first molar and extended to twice their resting length (12 mm). Nickel-titanium closing springs were chosen as several studies have shown that they produce a more consistent rate of space closure with sliding mechanics than elastics. [bib_ref] A randomized clinical trial to compare three methods of orthodontic space closure, Dixon [/bib_ref] [bib_ref] A comparison of the rate of space closure using a nickel-titanium spring..., Samuels [/bib_ref] [bib_ref] A clinical study of space closure with nickel-titanium closed coil springs and..., Samuels [/bib_ref] The springs were secured to a soldered brass hook on the distal of the upper lateral incisor with metal ligatures. The final length of the nickel-titanium coil spring was recorded [fig_ref] Figure 1: Standardized activation of nickel-titanium closing spring to approximately twice the resting length [/fig_ref]. The use of intermaxillary elastics was postponed until the end of the upper space closure stage of treatment.
The primary outcome measure was the amount of space closure in millimetres, in both quadrants of the maxillary arch, after 3 months. Once the 0.01960.025inch stainless steel archwire had been in situ for at least 6 weeks and before space closure commenced, archwires were removed and alginate impressions of the maxillary arch were taken (T0). The impressions were cast in white stone on-site and within the same day. All patients were recalled at 4 week intervals for 3 months, or until space closure was complete. At each subsequent visit, appliances were assessed for damage, additional maxillary arch impressions were made once the archwire had been removed and the nickel-titanium springs were reactivated to 12 mm. The space closure study was discontinued at the end of 3 months, or one appointment before space closure was complete, to avoid the possibility that the spaces were closed and tooth movement finished before the next adjustment appointment. The aim was to collect four sets of maxillary arch models for each patient (T0-T3) by the end of the trial period.
Prior to measurement the patient details and dates when the study models were taken were masked and each cast was given a randomly generated number by an orthodontist not involved in the study. The size of the two upper arch extraction spaces were measured in the randomly generated order by one operator (HW), who was unaware of the group allocation, using digital callipers and the method described by Dixon et al. [bib_ref] A randomized clinical trial to compare three methods of orthodontic space closure, Dixon [/bib_ref] After 2 weeks, 20 models were randomly selected, recoded in a new random order and the measurements repeated.
An a priori sample size calculation was undertaken based on data from the study by Dixon et al. [bib_ref] A randomized clinical trial to compare three methods of orthodontic space closure, Dixon [/bib_ref] They found a mean difference in space closure between active ligatures and nickel-titanium springs of 0.46 mm/month (SD: 0.86 mm). Using these data, we estimated that a sample size of 13 subjects in each group would be sufficient to detect a difference in the rate of space closure of 3 mm (SD: 2.58) over 3 months, with a power of 90% and significance level of 0.05. To account for a 15% drop-out rate, the sample size was increased to 15 participants per group.
# Statistical analysis
Once all the measurements had been undertaken the masking codes were broken and the data entered in a spreadsheet (Excel; Microsoft Corp., Redmond, WA, USA). The size of each remaining extraction space (in mm) measured on the models from time points T1, T2 and T3 was subtracted from the size of the remaining extraction space measured from the same site on the models from the preceding time point (i.e. T1-T0; T2-T1; T3-T2). This figure was then divided by the actual number of days that had elapsed between each study cast to obtain the amount of space closure per day. This was multiplied by 84 (28 days in 4 weeks multiplied by 3) to obtain a standard amount of space closure for each extraction site over three months.
The distribution of the data was examined using the Shapiro-Wilk test and found to be normal (P50.753). The repeatability of the measurements was assessed using an intra-class correlation coefficient (ICC) for random error and a paired t test for systematic error.
The null hypothesis that there was no difference between the three groups was tested using a one-way ANOVA. The dependent variable was the standardized amount of space closure over 3 months. The independent variable was group allocation. SPSS Statistics for Windows (v19 SPSS Inc., Chicago, IL, USA) was used for all statistical analyses.
# Results
The progression of participants through the trial is shown in [fig_ref] Figure 2: CONSORT diagram showing the flow of participants through the trial after 3... [/fig_ref]. A total of 45 patients were enrolled in the study, of which three were lost to follow-up (two relocated and one withdrew). In two patients, the extraction sites on both sides of the upper arch were closed within one visit and therefore no usable data were gathered. Of the remaining participants, the extraction sites on one side were closed within one visit in four patients, therefore data from only one side was included in the analysis. The baseline characteristics of the participants are shown in [fig_ref] Table 1: Baseline characteristics of the three groups of patients [/fig_ref]. The mean age at enrolment was 13.9 years (SD: 1 year 10 months), with 23 girls and 17 boys.
The repeatability of the space measurements was high with a mean difference between the first and second reading of 0.05 mm (SD: 0.24) and an intraclass correlation coefficient of 0.995, indicating a low level of random error. There was no evidence of systematic error (P50.199).
The amount of space closure between the right and left sides of the arch within individuals was examined using a paired t test and no significant difference was found (mean difference was 20.14 mm; SD: 1.22; P50.498); therefore, the mean of the two sides was used for hypothesis testing. The descriptive statistics for the standardized amount of space closure in 3 months are shown in . The amounts of space closure were very similar between the three groups and averaged approximately 1 mm/month; however, there was a wide variation in the rate of space closure between individuals from 0.10 mm/month in one participant to 1.80 mm/ month in another. The mean differences in space closure [fig_ref] Table 3: One-way ANOVA with a dependent variable of standardized amount of space closure... [/fig_ref]. The one-way ANOVA confirmed that the bracket/ligature combination had no significant effect on the amount of space closure over this period (P50.718).
# Discussion
This randomized controlled clinical trial did not find any significant differences in the average amount of space closure over 3 months between participants treated using three different bracket-ligature combinations. It would appear therefore, that factors other than bracketligature combinations determine the rate of orthodontic space closure.
These findings are consistent with previous prospective clinical trials that have found no differences in the rate of tooth movement between conventional and selfligating brackets during initial alignment, 13-16 en masse space closure using sliding mechanics 17 retraction of upper canine teeth 18 or overall treatment time in extraction cases. [bib_ref] Duration of treatment and occlusal outcome using Damon3 self-ligated and conventional orthodontic..., Dibiase [/bib_ref] Pandis and colleagues 20 did find more rapid alignment when using self-ligating brackets in patients with moderately crowded lower incisors compared to conventional brackets, but in patients with severe crowding there were no differences. Interestingly, Burrow 21 concluded that canine retraction was quicker within individuals when using a conventional bracket compared with a self-ligating bracket on the contralateral side. Two recent systematic reviews have found no convincing evidence of improved treatment efficiency when self-ligating brackets are used. [bib_ref] Systematic review of self-ligating brackets, Chen [/bib_ref] [bib_ref] Self-ligating brackets in orthodontics. A systematic review, Fleming [/bib_ref] The average rate of space closure in this study was 1mm per month, which is very similar to other space closure studies. [bib_ref] A randomized clinical trial to compare three methods of orthodontic space closure, Dixon [/bib_ref] [bib_ref] Self-ligating vs conventional twin brackets during en-masse space closure with sliding mechanics, Miles [/bib_ref] [bib_ref] Maxillary canine retraction with self-ligating and conventional brackets, Mezomo [/bib_ref] [bib_ref] Canine retraction rate with self-ligating brackets vs conventional edgewise brackets, Burrow [/bib_ref] [bib_ref] A clinical investigation of force delivery systems for orthodontic space closure, Nightingale [/bib_ref] Also consistent with other studies was the finding that there was a wide variation in the rates of tooth movement between individual participants. Pilon et al. [bib_ref] Magnitude of orthodontic forces and rate of bodily tooth movement. An experimental..., Pilon [/bib_ref] showed that despite all attempts to standardize the delivery of forces to the teeth of beagle dogs, the amount of tooth movement was much more dependent upon individual variation between dogs than on the force levels. They speculate that differences between 'slow movers' and 'fast movers' are due to variations in bone density, bone metabolism, and in particular turnover of the periodontal ligament, rather than treatment factors, such as force levels or bracket/ligature combinations.
We attempted to standardize the participant appointment intervals to every 4 weeks or 28 days, which is similar to other studies. [bib_ref] Clinical efficiency of nonconventional elastomeric ligatures in the canine retraction phase of..., Dholakia [/bib_ref] [bib_ref] Maxillary canine retraction with self-ligating and conventional brackets, Mezomo [/bib_ref] [bib_ref] Self-ligating vs conventional brackets in the treatment of mandibular crowding: a prospective..., Pandis [/bib_ref] Unfortunately, it was not possible for all patients to attend exactly every 28 days; therefore an average daily amount of space closure was calculated according to the dates the study models were taken. This was multiplied by 28 to achieve the standardized amount per month, then multiplied by three to find a standardized amount over 3 months. We realize that in clinical practice recall intervals might be longer; however, 4 weeks was chosen because shorter time periods might prove to be a burden to participants and their parents, whereas longer recall intervals might lead to inaccuracies in recording the rate of tooth movement, as the spaces would be completely closed before the patient returns for their subsequent appointment.
Space closure was undertaken using nickel-titanium coil springs as they have been shown to be an efficient method of tooth movement. [bib_ref] A randomized clinical trial to compare three methods of orthodontic space closure, Dixon [/bib_ref] [bib_ref] A comparison of the rate of space closure using a nickel-titanium spring..., Samuels [/bib_ref] [bib_ref] A clinical study of space closure with nickel-titanium closed coil springs and..., Samuels [/bib_ref] Sliding mechanics was carried out en masse, by including the whole of the labial segment (incisors and canines) against the premolars and molars and avoiding an initial canine retraction stage. En masse space closure represents common clinical practice and is usually required for patients treated with a pre-adjusted edgewise appliance and premolar extractions. The use of intermaxillary elastics was avoided until after space closure had been achieved, although Dixon and colleagues, 10 did not find this to be a significant factor explaining the rate of space closure. Another potential confounder could have been the number of broken appliances during the experimental period. There were eleven incidences of 'broken appliances' recorded in trial participants during the experimental period; however, only on four occasions were these considered relevant to space closure mechanics. As this number was small (4 out of 263 readings; 1.5%), we considered the effect on the results minimal.
We chose an inter-individual, parallel group design, similar to Dixon et al., [bib_ref] A randomized clinical trial to compare three methods of orthodontic space closure, Dixon [/bib_ref] as we did not consider the two sides of space closure to be mutually independent of each other because they involved the same archwire. Several studies investigating space closure or canine retraction have used an intra-individual, split mouth design. [bib_ref] Clinical efficiency of nonconventional elastomeric ligatures in the canine retraction phase of..., Dholakia [/bib_ref] [bib_ref] A comparison of the rate of space closure using a nickel-titanium spring..., Samuels [/bib_ref] [bib_ref] A clinical study of space closure with nickel-titanium closed coil springs and..., Samuels [/bib_ref] [bib_ref] Self-ligating vs conventional twin brackets during en-masse space closure with sliding mechanics, Miles [/bib_ref] [bib_ref] Maxillary canine retraction with self-ligating and conventional brackets, Mezomo [/bib_ref] [bib_ref] Canine retraction rate with self-ligating brackets vs conventional edgewise brackets, Burrow [/bib_ref] The advantage of an intra-individual design is that it might allow more precise comparison between techniques by removing the confounding factor of variability in the rate of tooth movement between individuals. Conversely, it could be argued that it would be unusual to use different methods of space closure on the two sides of an arch in the same individual, therefore this does not accurately reproduce orthodox clinical practice. It might also be possible that a split mouth design would introduce an additional confounder if the method of space closure on one side of the arch either increased or decreased the rate of tooth movement on the contra-lateral side. For these reasons we believe that an inter-individual, parallel group design to compare bracket/ligature combinations was appropriate. Although we found no statistical difference in the rate of space closure between the right and left sides of the arch, the sample size was not based on detecting this difference. Closer examination of the data revealed that when there were available data from both sides of the arch over half the readings (45 out of 87; 52%) demonstrated a 0.5 mm or greater difference in the amount of tooth movement between the sides (max 4.3 mm difference after 72 days). This would suggest that a parallel group study design would be more appropriate for research in this area in the future.
The method of data collection was similar to that of Mezomo et al. [bib_ref] Maxillary canine retraction with self-ligating and conventional brackets, Mezomo [/bib_ref] and Dholakia and Bhat. [bib_ref] Clinical efficiency of nonconventional elastomeric ligatures in the canine retraction phase of..., Dholakia [/bib_ref] Study models were taken immediately before space closure was started and at each 4-week adjustment appointment for three visits or until the both spaces were closed. This allowed a visit-by-visit assessment of space closure, but more importantly the records were suitably masked and anonymized prior to both the initial and repeat measurements to minimize assessment bias. Neither Mezomo et al. [bib_ref] Maxillary canine retraction with self-ligating and conventional brackets, Mezomo [/bib_ref] nor Dholakia and Bhat 4 explain whether they masked their models. Dixon and colleagues 10 collected data from masked study models taken before space closure was started and at four months or 'earlier if space closure was complete', but do not state what their recall appointment times were between 1 and 4 months. Miles 17 and Burrow 21 collected data from direct intra-oral measurements, the former using a digital calliper and the latter a flexible ruler. Although the investigators took several readings and averaged them, which would help to minimize random measurement error, neither describes how they adequately blinded the assessor as to group-side allocation.
We used data from a previous study [bib_ref] A randomized clinical trial to compare three methods of orthodontic space closure, Dixon [/bib_ref] to support an a priori sample size calculation to find a significant difference in the rate of space closure of 3 mm in 3 months. This was chosen as we considered it to be a reasonable length of time to detect a difference in the amount of space closure between the three groups if a difference did actually exist. It could be argued that 3 mm over 3 months is a large clinical difference to detect and a smaller difference would be more reasonable. Dixon et al. [bib_ref] A randomized clinical trial to compare three methods of orthodontic space closure, Dixon [/bib_ref] used a similar figure in their sample size calculation and estimated that an average participant in their trial with 2 mm of space left in any quadrant at the end of four months would have a reduced treatment time of 3.2 months if treated with a nickel-titanium closing coil compared with an active ligature. The value for the variability of space closure over 3 months (2.58 mm) was an estimate, as we were unable to find any data in the literature. It was derived by multiplying the standard deviation quoted by Dixon et al. [bib_ref] A randomized clinical trial to compare three methods of orthodontic space closure, Dixon [/bib_ref] for the variation in the monthly rate of space closure by three. This proved to be an overestimate of the variability in the rate of space closure over 3 months . When the actual standard deviation (1.2 mm) from the study is used in the calculation the sample size was sufficient to detect a difference of 1.4 mm over 3 months with the same power and significance level.
We found a higher proportion of participants in our trial achieved space closure on one or both sides before the end of the 3-month trial period (22 patients, 52%) compared to other studies. Miles [bib_ref] Self-ligating vs conventional twin brackets during en-masse space closure with sliding mechanics, Miles [/bib_ref] reports that in two out of 15 patients who completed his trial (13%) the spaces closed during the alignment phase and were therefore excluded from the analysis. Dixon et al. [bib_ref] A randomized clinical trial to compare three methods of orthodontic space closure, Dixon [/bib_ref] found that approximately one-third of the quadrants in their study were closed before the end of the 4-month experimental period. Our data would suggest that even if we had found a significant difference of 3 mm in a 3month period between participant groups that this would equate to a saving of one visit or 4-6 weeks over the course of a 2-year treatment. It would be interesting to determine if patients consider this to be clinically significant.
Another criticism of this study might be that the sample size was too small to detect a significant difference between the bracket/archwire combinations; however, the descriptive data showed that there were very small mean differences in the amount of space closure between the three groups after 3 months with wide overlap of the confidence intervals. Based on these data, it is very unlikely that a study with a considerably larger sample size would detect a significant difference and suggests that the hypothesis of reducing static friction by modifying the bracket/ligature interface in order to increase the rate of tooth movement and hence reduce the length of orthodontic treatment, is questionable. Laboratory investigations indicate that archwire binding and notching have a more significant effect on any resistance to movement than static or dynamic friction. [bib_ref] Effects of ligation type and method on the resistance to sliding of..., Thorstenson [/bib_ref] The effects of other factors present in the mouth, such as salivary lubrication, shock absorption of the periodontal ligament and stress-breaking perturbations during mastication have also not been considered. Animal and clinical studies demonstrate that the largest factor in determining the rate of tooth movement is probably the individual patient response to any applied force.
# Conclusions
N No differences were found in the amount of space closure between three different bracket/archwire combinations.
N The largest factor in determining the rate of tooth movement is probably the individual patient response to any applied force.
[fig] Figure 1: Standardized activation of nickel-titanium closing spring to approximately twice the resting length(12 mm) [/fig]
[fig] Figure 2: CONSORT diagram showing the flow of participants through the trial after 3 months between the three groups were very small, ranging from 0.1 to 0.3 mm and there was considerable overlap in the confidence intervals for the differences between the groups [/fig]
[table] Table 1: Baseline characteristics of the three groups of patients. [/table]
[table] Table 3: One-way ANOVA with a dependent variable of standardized amount of space closure over 3 months and independent variable of bracket/ligature combination. The descriptives are for the differences between groups. [/table]
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Conservative Treatments Frequently Used for Chronic Pain Patients in Clinical Practice: A Literature Review
# Introduction and background
The most current definition of pain by the International Association for the Study of Pain (IASP) is "an unpleasant sensory and emotional experience associated with, or resembling that associated with, actual or potential tissue damage". Chronic pain is defined as pain suffered for longer than three months or after complete healing. It may arise as a consequence of tissue damage, inflammation, and may have no identified cause. It has been reported that around 20% of the adult population suffers from chronic pain and in 8% of the individuals, the pain is so severe that it interferes with life or work activities. Such chronic pain often brings about psychiatric disorders such as depression and anxiety. Chronic pain is not a simple extension of acute pain and can occur spontaneously without stimulus, while its degree is not proportional to that of the original damage. Such chronic pain can cause neural plasticity or sensitization of the nervous system in the peripheral nerve, spinal cord, and brain. In other words, it induces alterations to the nervous system. Thus, chronic pain should be considered not as a mere symptom of disease, but as a disease in itself of the nervous system. To date, various methods have been applied to control chronic pain including exercise, physical therapy, medication, procedures, surgery, and psychotherapy. In many cases, the symptoms of chronic pain are not fully resolved even with treatment, and a single mode of treatment often fails to control pain, necessitating the need for multiple treatment methods. This article aims to outline the conservative treatment methods often used in clinical practice to control chronic pain.
## Review 1. medication
## Anticonvulsants
Anticonvulsants are drugs used for the inhibition of seizures, which are also effective for neuropathic pain control. Anticonvulsants control pain by inhibiting abnormal ectopic excitation in the injured nerve at a dose that does not inhibit the conduction of normal nerves. Since gabapentin and pregabalin are effective for pain control and cause only minor adverse reactions, they are often used in clinical practice. While both drugs have γ-aminobutyric acid (GABA)-like structures, they have no GABA-related activities and have no efficacy on the reuptake or metabolism of GABA. They bind to the α2-δ subunit of the voltage-dependent calcium channels at synapses, which reduces the release of pain-related neurotransmitters. Since these drugs are eliminated through the kidney, patients with a reduced renal function are likely to have adverse reactions such as severe dizziness or sedation, and the dose would therefore need to be reduced.
## Nonsteroidal anti-inflammatory drugs
Nonsteroidal anti-inflammatory drugs (NSAIDs) suppress inflammatory actions by the inhibition of cyclooxygenase (COX)-1/2, resulting in an analgesic effect. While they are effective for the control of chronic pain related to higher inflammation, such as osteoarthritis and rheumatoid arthritis, long-term use is not advisable due to adverse reactions such as gastrointestinal bleeding and nephrotoxicity.
## Opioids
Opioids include codeine, hydrocodone, and tramadol (not a true classic opioid with a week opioid agonist with serotonin-norepinephrine reuptake inhibitor properties). They induce an analgesic effect and adverse reactions through interactions with various opiate receptors. Opioids are effective for persistent and dull pain rather than intermittent and sharp pain. Adverse reactions include nausea, vomiting, sedation, dizziness, and constipation. However, tramadol is not a true classic opioid.
## Antidepressants
In addition to the treatment of mood disorders, antidepressants also have efficacy for pain control. They inhibit the reuptake of neurotransmitters, such as serotonin or norepinephrine, and block potassium channels in the descending inhibitory pain pathway of the brain and spinal cord, which prevents the ectopic discharge of the injured nerve, leading to an analgesic action. Antidepressants that are frequently used for the treatment of chronic pain in clinical practice include tricyclic antidepressants, selective serotonin reuptake inhibitors, and serotonin-norepinephrine reuptake inhibitors. These drugs are known to be effective mainly for neuropathy as well as tension headache, migraine, and fibromyalgia syndrome. Adverse reactions of antidepressants include cardioplegia, arrhythmia, orthostatic hypotension, xerostomia, constipation, ischuria, vomiting, nausea, dizziness, and sedation, and are therefore prescribed at low doses initially, followed by a gradual increase.
## Procedures
## Injection of steroids and local anesthetics
Intervertebral disc herniation or stenosis of the spine can cause inflammation or compression of the vertebral nerve, which often leads to chronic pain. In addition, compression of peripheral nerves, as in carpal tunnel syndrome, tardy ulnar neuropathy, meralgia paresthetica, and tarsal tunnel syndrome, can be accompanied by neurogenic pain, often leading to chronic pain. A nerve blockade with a steroid and local anesthetics can effectively control such pain. In addition, an injection of a steroid and local anesthetic into the joint can mitigate joint-origin pain. This treatment has been known to block the transmission in nociceptive C-fiber and reduce ectopic discharge. Moreover, steroids can control pain through the excitatory blockage of the peripheral nerve via the functional inhibition of inflammatory mediators. Nerve blocks with steroids also inhibit the synthesis of various mediators of inflammation and reduce the inflammation of nerves induced by mechanical compression. However, when nerves are severely compressed, injured, or damaged, the effectiveness of a nerve block with steroids and local anesthetics is reduced. Recently, fluoroscopy-or ultrasound-guided injections have been utilized to accurately inject steroids into the site of pain. Since repeated injections of steroids affect the secretion of adrenocortical hormones, besides causing adverse reactions such as elevation of blood sugar levels, elevated blood pressure, and osteoporosis, the number of administration must be controlled.
## Pulsed radiofrequency
Unlike conventional radiofrequency (RF) that controls pain through the ablation of nerves at a high temperature, pulsed radiofrequency (PRF) uses a needle tip under 42 °C by adding intervals between the electric stimuli to prevent the ablation of tissues. PRF is applied not only for neurogenic pain, but also for various chronic pain disorders including muscle pain, joint pain, and discogenic pain. While the mechanism of PRF in the control of chronic pain is yet to be understood, PRF is thought to alter pain transmission by inhibiting pain impulse propagation. When PRF was applied to the dorsal root ganglia in a rat model of a herniated lumbar disc, microglial activity, involved in the generation and amplification of chronic pain, was reduced in the dorsal horn. Because microglia release several cytokines and chemokines that are associated with progression to chronic pain, the down-regulation of their activities may control pain. Additionally, PRF stimulation may cause microscopic damage to unmyelinated C-fibers that transfer pain sensation. In 2016, Lee et al. compared the effects of PRF and a nerve root block with steroids on radicular pain due to a herniated disc. They recruited 38 patients and randomly allocated them to PRF and steroid groups. After both procedures, radicular pain was significantly reduced, and it lasted for at least 12 weeks. During the 12-week period after each procedure, their pain-reducing effects were also similar. In 2020, Lee et al. performed PRF on cervical nerve roots in 49 patients who did not show a positive response to a nerve root block with a steroid, and they reported a significant pain reduction. Therefore, PRF may be effective in patients who need frequent injections with steroids and in those who do not experience pain reduction after injection with steroids.
## Repetitive transcranial magnetic stimulation
Repetitive transcranial magnetic stimulation (rTMS) induces electric currents in the brain using high-frequency stimuli that have a short duration through a strong magnetic field. At least 1,000 impulses below 1 ms are applied. It has been reported that rTMS is effective not only for neurogenic pain, but also for pain attributable to fibromyalgia syndrome, lumbar pain, and shoulder pain. The primary motor cortex is the main target of stimulation. Previous functional magnetic resonance imaging studies have demonstrated that rTMS on the primary motor cortex induces changes in the activity of cortical and subcortical structures related to pain processing and modulation. Furthermore, the application of rTMS on the primary motor cortex may influence the endogenous opioid system. However, most effective rTMS stimulation modes or sites, according to the types of chronic pain, have not been thoroughly elucidated. Further studies should clarify these.
## Prolotherapy
Incomplete healing or partial damage to ligaments can result in secondary laxity and lability of the joint regulated by the ligament, ultimately resulting in chronic pain. Prolotherapy refers to a treatment for such chronic pain in which a small amount of stimulant is injected into the ligament or tendon enthesis, inducing regeneration of new cells via inflammatory responses. Stimulants are injected into ligaments attached to the bone or tendon enthesis, leading to inflammation, which subsequently induces the healing process. In other words, inflammatory mediators stimulate the secretion of growth factors that are directly helpful for healing, recruiting various cells, of which fibroblasts are the most important for the regeneration of the musculoskeletal system. Regarding the treatment mechanism, these cells not only heal by inflammation but also actively produce collagen, inducing the regeneration of injured ligaments without healing, as well as tendon enthesis, which ultimately strengthens ligaments and tendon enthesis, leading to stability for pain control.
Usually, dextrose is used alone or in combination with other substances such as lidocaine or bupivacaine. Since it has been reported that at least 10% of dextrose is needed to induce inflammation, dextrose should be prepared at a concentration higher than 10%. However, concentrations of dextrose above 25% can cause problems such as the induction of severe pain or damage to proximal normal tissues; therefore, higher concentrations should be avoided. Thus, 10-25% of dextrose is generally used in prolotherapy.
## Exercise
Chronic pain is likely to be accompanied by muscle atrophy, weakness, or joint contracture, which can be prevented by exercise. Chronic pain can also cause fatigue, inertia, and depression. Exercise increases the levels of adrenocortical hormones, cortisol, and catecholamine, which in turn produce beta-endorphins, resulting in reducing the sensation of pain. Furthermore, exercise itself can be the most important treatment for some diseases. For example, muscle strengthening is the most fundamental treatment method for patellofemoral pain syndrome, attributable to the weakness of the vastus medialis. As for lateral and medial epicondylitis, it is important to follow exercise regimens that strengthen the wrist extensor and the wrist flexor tendon, respectively. Also, in patients with fibromyalgia, exercise was reported to simulate brain regions involving in descending pain inhibition. Many previous studies have reported that exercise can reduce the severity of pain, enhance physical function, and improve psychological function and quality of life. However, these positive effects of exercise were not found in all studies. This inconsistency could be due to the application of different types of exercise in each study and the quality of the studies. Therefore, further studies are needed on this topic.
# Conclusions
Various mechanisms are involved in the onset of chronic pain, even when the cause is identical, and various types of pain can be induced. Thus, effective treatment methods are dependent on each individual's needs, and in most cases, chronic pain can be more effectively controlled by a combination of various treatment methods. In addition to the aforementioned treatment methods to reduce chronic pain, various other methods have also been attempted, and effective outcomes have been consequently reported. Clinicians should be aware of currently employed treatment methods for each type of chronic pain to most effectively treat patients. Efforts should also be made to update the knowledge of clinicians about the innovative treatment methods that are being developed, followed by their application in patients.
## Additional information
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MetaCRAM: an integrated pipeline for metagenomic taxonomy identification and compression
Background: Metagenomics is a genomics research discipline devoted to the study of microbial communities in environmental samples and human and animal organs and tissues. Sequenced metagenomic samples usually comprise reads from a large number of different bacterial communities and hence tend to result in large file sizes, typically ranging between 1-10 GB. This leads to challenges in analyzing, transferring and storing metagenomic data. In order to overcome these data processing issues, we introduce MetaCRAM, the first de novo, parallelized software suite specialized for FASTA and FASTQ format metagenomic read processing and lossless compression. Results: MetaCRAM integrates algorithms for taxonomy identification and assembly, and introduces parallel execution methods; furthermore, it enables genome reference selection and CRAM based compression. MetaCRAM also uses novel reference-based compression methods designed through extensive studies of integer compression techniques and through fitting of empirical distributions of metagenomic read-reference positions. MetaCRAM is a lossless method compatible with standard CRAM formats, and it allows for fast selection of relevant files in the compressed domain via maintenance of taxonomy information. The performance of MetaCRAM as a stand-alone compression platform was evaluated on various metagenomic samples from the NCBI Sequence Read Archive, suggesting 2-to 4-fold compression ratio improvements compared to gzip. On average, the compressed file sizes were 2-13 percent of the original raw metagenomic file sizes. Conclusions: We described the first architecture for reference-based, lossless compression of metagenomic data. The compression scheme proposed offers significantly improved compression ratios as compared to off-the-shelf methods such as zip programs. Furthermore, it enables running different components in parallel and it provides the user with taxonomic and assembly information generated during execution of the compression pipeline. Availability: The MetaCRAM software is freely available at http://web.engr.illinois.edu/~mkim158/metacram.html. The website also contains a README file and other relevant instructions for running the code. Note that to run the code one needs a minimum of 16 GB of RAM. In addition, virtual box is set up on a 4GB RAM machine for users to run a simple demonstration.
# Background
Metagenomics is an emerging discipline focused on genomic studies of complex microorganismal population. In particular, metagenomics enables a range of analyses pertaining to species composition, the properties of the species and their genes as well as their influence on the host organism or the environment. As the interactions between microbial populations and their hosts plays an important role in the development and functionality of the host, metagenomics is becoming an increasingly important research area in biology, environmental and medical sciences. As an example, the National Institute of Health (NIH) recently initiated a far-reaching Human Microbiome Project [bib_ref] The nih human microbiome project, Peterson [/bib_ref] which has the aim to identify species living at different sites of the human body (in particular, the gut and skin [bib_ref] Skin microbiome: genomics-based insights into the diversity and role of skin microbes, Kong [/bib_ref] , observe their roles in regulating metabolism and digestion, and evaluate their influence on the immune system. The findings of such studies may have important impacts on our understanding of the influence of microbials on an individual's health and disease, and hence aid in developing personalized medicine approaches. Another example is the Sorcerer II Global Ocean Sampling Expedition [bib_ref] The sorcerer ii global ocean sampling expedition: northwest atlantic through eastern tropical..., Rusch [/bib_ref] , led by the Craig Venter Institute, the purpose of which is to study microorganisms that live in the ocean and influence/maintain the fragile equilibrium of this ecosystem.
There are many challenges in metagenomic data analysis. Unlike classical genomic samples, metagenomic samples comprise many diverse organisms, the majority of which is usually unknown. Furthermore, due to low sequencing depth, most widely used assembly methods -in particular, those based on de Bruijn graphsoften fail to produce quality results and it remains a challenge to develop accurate and sensitive meta-assemblers. These and other issues are further exacerbated by the very large file size of the samples and their ever increasing number. Nevertheless, many algorithmic methods have been developed to facilitate some aspects of microbial population analysis: examples include MEGAN (MEta Genome ANalyzer) [bib_ref] Megan analysis of metagenomic data, Huson [/bib_ref] , a widely used tool that allows for an integrative analysis of metagenomic, metatranscriptomic, metaproteomic, and rRNA data; and PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) [bib_ref] Predictive functional profiling of microbial communities using 16s rrna marker gene sequences, Langille [/bib_ref] , developed to predict metagenome functional contents from 16S rRNA marker gene sequences. Although suitable for taxonomic and functional analysis of data, neither MEGAN nor PICRUSt involve a data compression component, as is to be expected from highly specialized analytic software.
In parallel, a wide range of software solutions have been developed to efficiently compress classical genomic data (a comprehensive survey of the state-of-the-art techniques may be found in [bib_ref] Data compression for sequencing data, Deorowicz [/bib_ref]. Specialized methods for compressing whole genomes have been reported in [bib_ref] Dna sequence compression using the burrows-wheeler transform, Adjeroh [/bib_ref] [bib_ref] Protein is incompressible, Nevill-Manning [/bib_ref] [bib_ref] Dnacompress: fast and effective dna sequence compression, Chen [/bib_ref] , building upon methods such as modified Lempel-Ziv encoding and the Burrows-Wheeler transform. Compression of reads is achieved by mapping the reads to reference genomes and encoding only the differences between the reference and the read; or, in a de novo fashion that does not rely on references and uses classical sequence compression methods. Quip [bib_ref] Compression of next-generation sequencing reads aided by highly efficient de novo assembly, Jones [/bib_ref] and CRAMare two of the best known reference-based compression algorithms, whereas ReCoil [bib_ref] Recoil-an algorithm for compression of extremely large datasets of dna data, Yanovsky [/bib_ref] , SCALCE [bib_ref] Scalce: boosting sequence compression algorithms using locally consistent encoding, Hach [/bib_ref] , MFCompress [bib_ref] Mfcompress: a compression tool for fasta and multi-fasta data, Pinho [/bib_ref] , and the NCBI Sequence Read Archive method compress data without the use of reference genomes. Reference-based algorithms in general achieve better compression ratios than reference-free algorithms by exploiting the similarity between some predetermined reference and the newly sequenced reads. Unfortunately, none of the current reference-based method can be successfully applied to metagenomic data, due to the inherent lack of "good" or known reference genomes. Hence, the only means for compressing metagenomic FASTA and FASTQ files is through the use of de novo compression methods.
As a solution to the metagenomic big data problem, we introduce MetaCRAM, the first de novo, parallel, CRAMlike software specialized for FASTA-format metagenomic read compression, which in addition provides taxonomy identification, alignment and assembly information. This information primarily facilitates compression, but also allows for fast searching of the data in the compressive domain and for basic metagenomic analysis. The gist of the classification method is to use a taxonomy identification tool -in this case, Kraken [bib_ref] Kraken: ultrafast metagenomic sequence classification using exact alignments, Wood [/bib_ref] which can accurately identify a sufficiently large number of organisms from a metagenomic mix. By aligning the reads to the identified reference genomes of organisms via Bowtie2 [bib_ref] Fast gapped-read alignment with bowtie 2, Langmead [/bib_ref] , one can perform efficient lossless reference-based compression via the CRAM suite. Those reads not aligned to any of the references can be assembled into contigs through existing metagenome assembly software algorithms, such as Velvet [bib_ref] Velvet: algorithms for de novo short read assembly using de bruijn graphs, Zerbino [/bib_ref] or IDBA-UD [bib_ref] Idba-ud: a de novo assembler for single-cell and metagenomic sequencing data with..., Peng [/bib_ref] ; sufficiently long contigs can subsequently be used to identify additional references through BLAST (Basic Local Alignment Search Tool) [bib_ref] Basic local alignment search tool, Altschul [/bib_ref]. The reads aligned to references are compressed into the standard CRAM format, using three different integer encoding methods, Huffman [bib_ref] A method for the construction of minimum redundancy codes, Huffman [/bib_ref] , Golomb [bib_ref] Run-length encodings, Golomb [/bib_ref] , and Extended Golomb encoding [bib_ref] Extended golomb code for integer representation, Somasundaram [/bib_ref].
MetaCRAM is an automated software with many options that accommodate different user preferences, and it is compatible with the standard CRAM and SAMtools data format. In addition, its default operational mode is lossless, although additional savings are possible if one opts for discarding read ID information. We report on both the lossless and "lossy" techniques in the "Methods" Section. MetaCRAM also separates the read compression process from the quality score compression technique, as the former technique is by now well understood while the latter is subject to constant changes due to different quality score formats in sequencing technologies. These changes may be attributed to increasing qualities of reads and changes in the correlations of the score values which depend on the sequencing platform. For quality score compression, the recommended method is QualComp [bib_ref] Qualcomp: a new lossy compressor for quality scores based on rate distortion..., Ochoa [/bib_ref].
MetaCRAM offers significant compression ratio improvements when compared to standard bzip and gzip methods, and methods that directly compress raw reads. These improvements range from 2-4 fold file size reductions, which leads to large storage cost reductions. Furthermore, although MetaCRAM has a relatively long compression phase, decompression may be performed in a matter of minutes. This makes the method suitable for both real time and archival applications.
The paper is organized as follows. The "Results" Section contains an in-depth performance analysis of MetaCRAM with respect to processing and retrieval time, and achievable compression ratios. The "Discussion" Section describes the advantages of using MetaCRAM for data compression compared to other general-purpose methods, and describes directions for future algorithmic improvements. The "Methods" Section contains detailed information about the methodology behind the MetaCRAM algorithmic blocks and it also outlines the way constituent algorithms are integrated and their purposes in the pipeline.
# Results
The block diagram of the MetaCRAM algorithm is given in , and the operation of the algorithm may be succinctly explained as follows. The first step is to identify suitable references for compression, which is achieved by identifying dominant taxonomies in the sample. The number of references is chosen based on cut-off abundance thresholds, which themselves are chosen using several criteria that trade-off compression ratio and compression time. Once the references are chosen, the raw reads are aligned to their closest references and the starting positions of the reads are statistically analyzed to determine the best integer compression method to be used for their encoding. Furthermore, reads that do not align sufficiently well with any of the chosen references are assembled using IDBA_UD, and the contig outputs of the assembler are used to identify additional references via BLAST search. Reads not matched with any references after multiple iterations of the above procedure are compressed independently with the MFCompress suite. The results associated with each of the described processing stages are discussed in the next subsections. Note that here and throughout the paper, we use standard terms in genomics and bioinformatics without explanations.
We tested MetaCRAM as a stand-alone platform and compared it to MFCompress, a recently developed software suite specialized for FASTA files, and bzip2 and gzip, standard general purpose compression tools (available at http://www.bzip.org). Other software tools for compression of sequencing data such as SCALCE and Quip, and SAMZIP [bib_ref] Improving transmission efficiency of large sequence alignment/map (sam) files, Sakib [/bib_ref] and SlimGene [bib_ref] Compressing genomic sequence fragments using slimgene, Kozanitis [/bib_ref] , were not As already pointed out, MetaCRAM does not directly process FASTQ file formats for multiple reasons: 1) the quality of sequencers are improving significantly, reaching the point where quality scores may contain very little information actually used during analysis; 2) reads with low quality scores are usually discarded and not included in metagenomics analysis -only high quality sequences are kept; 3) there exist software tools such as QualComp [bib_ref] Qualcomp: a new lossy compressor for quality scores based on rate distortion..., Ochoa [/bib_ref] , specifically designed for compressing quality scores that users can run independently along with MetaCRAM.
## Taxonomy identification and reference genome selection
As the first step of our analysis, we compared two metagenomic taxonomy identification programs, Kraken and MetaPhyler in terms of computation time and identification accuracy on synthetic data, as it is impossible to test the accuracy of taxonomy identification on real biological datasets. For this purpose, we created mixtures of reads from 15 species, listed in the Additional file 1. The two Illumina paired-end read files were created by MetaSim [bib_ref] Metasim: a sequencing simulator for genomics and metagenomics, Richter [/bib_ref] with 1 % error rate, and they amounted to a file of size 6.7 GB. Kraken finished its processing task in 22 min and successfully identified all species within the top 50 most abundant taxons. On the other hand, MetaPhyler ran for 182 min and failed to identify Acetobacterium woodii and Haloterrigena turkmenica at the genus level. This example illustrates a general trend in our comparative findings, and we therefore adopted Kraken as a default taxonomy retrieval tool for MetaCRAM.
When deciding how to choose references for compression, one of the key questions is to decide which outputs of the Kraken taxonomy identification tool are relevant. Recall that Kraken reports the species identified according to the number of reads matched to their genomes. The most logical approach to this problem is hence to choose a threshold for the abundance values of reads representing different bacterial species, and only use sequences of species with high abundance as compression references. Unfortunately, the choice for the optimal threshold value is unclear and it may differ from one dataset to another; at the same time, the threshold is a key parameter that determines the overall compression ratio -choosing too few references may lead to poor compression due to the lack of quality alignments, while choosing too many references may reduce the compression ratio due to the existence of many pointers to the reference files. In addition, if we allow too many references, we sacrifice computation time for the same final alignment rate. It is therefore important to test the impact of the threshold choice on the resulting number of selected reference genomes.
In [fig_ref] Table 1: Analysis of the influence of different threshold values on reference genome selection... [/fig_ref] , we listed our comparison results for all five datasets studied, using two threshold values: 75 (high) and 10 (low). For these two choices, the results are colored gray and white, respectively. We observe that we get slightly worse compression ratios if we select too few references, as may be seen for the files ERR321482 and ERR532393. Still, the processing time is significantly smaller when using fewer references, leading to 30 to 80 minutes of savings in real time. It is worth to point out that this result may also be due to the different qualities of internal hard drives: for example, the columns in gray were obtained running the code on Seagate Barracuda ST3000, while the results listed in white were obtained via testing on Western Digital NAS. Columns in bold represent a threshold of 75 species, while the columns not bolded correspond to a cutoff of 10 species. The results are shown for MetaCRAM-Huffman. "Align. %" refers to the alignment rates for the first and second round, and "No. files" refers to the number of reference genome files selected in the first and second iteration. Processing times are recorded row by row denoting real, user, and system time in order Many of the most abundant references may be from the same genus, and this may potentially lead to the problem of multiple alignment due to subspecies redundancy. The almost negligible effect of the number of reference genomes on alignment rate implies that combining them to remove the redundancy would improve computational efficiency, as suggested in. Nevertheless, extensive computer simulations reveal that the loss due to multiple alignment is negligible whenever we choose up to 75-100 references. Therefore, our recommendation is to use, as a rule of thumb, the threshold 75 in order to achieve the best possible compression ratio and at the same time provide a more complete list of genomic references for further analysis.
# Compression performance analysis
Our chosen comparison quality criteria include the compression ratio (i.e., the ratio of the uncompressed file and the compressed file size), as well as the compression and decompression time, as measured on an affordable general purpose computing platform: Intel Core i5-3470 CPU at 3.2 GHz, with a 16 GB RAM. We present test results for five datasets: ERR321482, SRR359032, ERR532393, SRR1450398, and SRR062462, including metagenomic samples as diverse as a human gut microbiome or a Richmond Mine biofilm sample, retrieved from the NCBI Sequence Read Archive [bib_ref] The sequence read archive, Leinonen [/bib_ref]. Additional file 2 contains detailed descriptions of the datasets tested.
The comparison results of compression ratios among six software suites are given in [fig_ref] Table 2: Comparison of compression ratios of six software suites [/fig_ref] and [fig_ref] Figure 2: Compression ratio [/fig_ref]. The methods compared include three different modes of MetaCRAM, termed Huffman, Golomb and Extended Golomb MetaCRAM. These three techniques differ from each other with respect to the integer compression scheme used. The schemes will be described in detail in the next sections, although we remark that the three methods are chosen to illustrate various compression ratio and decompression time trade-offs.
The result indicates that MetaCRAM using Huffman integer encoding method improves compression ratios of the classical gzip algorithm 2-3 fold on average. For example, MetaCRAM reduces the file size of SRR062462 to only 2 % of the original file size. Observe that MetaCRAM also offers additional features that go beyond compression only, such as taxonomy identification and assembly. Users have the options to retrieve the alignment rate, list of reference genomes, contig files, and alignment information in SAM format. This list produced by MetaCRAM may be stored with very small storage overhead and then used for quick identification of files based on their taxonomic content, which allows for selection in the compressive domain. Information regarding gene profiles was not included in the pipeline output, as gene analysis does not directly contribute to the quality of the compression algorithm.
In the listed results, the column named "Qual Value (MB)" provides the estimated size of the quality scores for each file, after alignment to references found by Kraken. In our implementation, we replaced these scores with a single "*" symbol per read and also removed the redundancy in read IDs. The result shows that these two options provide better ratios than the default ratio, as shown in [fig_ref] Table 2: Comparison of compression ratios of six software suites [/fig_ref] column "MCH2". However, since read IDs may be needed for analysis of some dataset, we also report results for the default "MCH1" mode which does not dispose of ID tags.
In terms of the processing time shown in [fig_ref] Table 3: Comparison of processing [/fig_ref] , the MetaCRAM suite is at a clear disadvantage, with processing time 150-fold slower than bzip2 in the worst case. [fig_ref] Figure 3: Average Runtime of Each Stage of MetaCRAM [/fig_ref] presents the average runtime of each stage for all five datasets tested, and illustrates that assembly, alignment, and BLAST search are computationally demanding, accounting for 62 percentage of the total time. This implies that removing the second and subsequent assembly rounds of MetaCRAM reduces the processing time significantly, at the cost of a smaller compression ratio. [fig_ref] Table 4: Comparison of compressed file sizes of MetaCRAM-Huffman using 2 rounds and 1... [/fig_ref] compares the compression ratios of MetaCRAM with one round and with two rounds of reference discovery, and indicates that removing the assembly, alignment and BLAST steps adds 1-6 MB to the compressed file size. Thus, the user has an option to skip the second round in order to expedite the processing time. Likewise, [fig_ref] Table 5: Comparison of retrieval [/fig_ref] illustrates that the retrieval time of MetaCRAM is longer than that of bzip2, gzip, and MFCompress, but still highly efficient. In practice, the processing time is not as relevant as the retrieval time, as compression is performed once while retrieval is performed multiple times. For long term archival of data, MetaCRAM is clearly the algorithm of choice since the compression ratio, rather than processing or retrieval time, is the most important quality criteria.
We also remark on the impact of different integer encoding methods on the compression ratio. Huffman, Golomb, and extended Golomb codes all have their advantages and disadvantages. For the tested datasets, Huffman clearly achieves the best ratio, as it represents the optimal compression method, whereas Golomb and extended Golomb compression improve the real and system time as a result of computation efficiency. However, the parallel implementation of MetaCRAM makes the comparison of processing time of the three methods slightly biased: for example, if we perform compression while performing assembly, compression will take much more time than compressing while running an alignment algorithm. As the processing and retrieval time is not consistent among the three methods, we recommend using Huffman coding for archival storage.
# Discussion
In what follows, we comment on a number of useful properties of the MetaCRAM program, including compatibility, losslessness, partial assembly results and compressive computing. Compatibility. MetaCRAM uses well established and widely tested genomic analysis tools, and it also follows the standard genomic data compression format CRAM, hence making the results of downstream analysis compatible with a current standard for genomic compression.
Lossless compression principle. By its very nature, MetaCRAM is a lossless compression scheme as it encodes the differential information between the reference and the metagenomic reads in a 100 % accurate fashion. Nevertheless, we enabled a feature that allow for some partial loss of information, such as the read ID tags. It is left to the discretion of the user to choose suitable options.
CRAM versus MFCompress. MFCompress achieves good compression ratios when compressing highly redundant reads. MetaCRAM consistently achieves a rate proportional to the alignment rate because it only encodes the small difference between the reference genome and the read. As more microbial genome become available, MetaCRAM will most likely offer higher compression For short hand notation, we used"MCH-2rounds" = MetaCRAM-Huffman with 2 rounds, "MCH-1round" = MetaCRAM-Huffman with 1 round. We also used the shortcut "MFComp" = MFCompress and "Align. %" refers to the percentage of reads aligned during 2 rounds and 1 round, respectively, for MCH-2rounds and MCH-1round ratio than other tools in general. Note that only on one data file -SRR359032 -did MFCompress achieve better compression ratios than MetaCRAM, most likely due to the redundancy issues previously mentioned. Metagenomic assembly. Metagenomic assembly is a challenging task, and there is a widely accepted belief that it is frequently impossible to perform meaningful assembly on mixture genomes containing species from related genomes. Nevertheless, we are using assembly mostly as a means for identifications, but at the same time its output provides useful contigs for gene transfer analysis and discovery. In the case that assembly fails on a dataset, we suggest skipping the assembly step so as to trade off computation time with discovery of new reference genomes and contigs.
Compressive computing. There has been an effort towards computing in the compressed domain, in order to eliminate the need for persistne compression and decompression time when all one needs to perform is simple alignment [bib_ref] Compressive genomics, Loh [/bib_ref]. Similarly, MetaCRAM offers easy retrieval and selection based on the list of references stored as an option. For example, suppose we perform MetaCRAM on all available human gut metagenome data. If we want to analyze the datasets with a concentration of Escherichia coli, we avoid sacrificing retrieval time by quickly scanning the list of reference files and only retrieving the datasets with E. coli.
# Methods
## Pre-processing
MetaCRAM accepts both unpaired and paired-end reads. If paired-end reads are given as an input to MetaCRAM, then the first preprocessing step is to append the read IDs with a "_1" or a "_2" indicating that the read came from the first or second mate, respectively. Another preprocessing step includes filtering out the quality scores in case that the input file is in FASTQ format. This filtering process allows for using new and emerging quality score compression methods without constantly updating the MetaCRAM platform. Note that the paired end labeling is done automatically, while filtering can be implemented outside the integrated pipeline by the user, based on his/her requirements for quality score lossy or lossless compression goals.
MetaCRAM uses as a default FASTA files that do not contain quality values, in which case the resulting SAM file contains the symbol "I" repeated as many times as the length of the sequence. These symbols amount to about 100 bytes per read, and this overhead increases proportionally to the number of reads [fig_ref] Table 2: Comparison of compression ratios of six software suites [/fig_ref] of the "Results" Section illustrates the amount of storage space that data quality scores occupy in each dataset, ranging from 153 MB to 3.4 GB). In order to reduce the size of this unnecessary field, MetaCRAM replaces the sequence of "I"s with a single symbol "*", complying with the standard SAM format. Likewise, read IDs are highly repetitive in nature: for instance, every read ID starts with the data name such as "SRR359032. ", followed by its unique read number. Rather than repeating the data name for every read, we simply store it once, and append it when performing decompression. Both versions of MetaCRAM -one incorporating these two options -and another one without the described features are available to the user. The former version of the methods requires a slightly longer compression and decompression time.
## Taxonomy identification
Given the labeled read sequences of a metagenomic sample, the first step is to identify the mixture of species present in the sample. There are several taxonomy identification methods currently in use: the authors of [bib_ref] Characterization of microbial diversity by determining terminal restriction fragment length polymorphisms of..., Liu [/bib_ref] proposes to use the 16S rRNA regions for bacterial genome identification, MetaPhyler [bib_ref] Taxonomic profiling for metagenomic sequences, Liu [/bib_ref] scans for unique markers exceeding length 20 and provides a taxonomy level as specific as the genus. On the other hand, a new taxonomy identification software known as Kraken [bib_ref] Kraken: ultrafast metagenomic sequence classification using exact alignments, Wood [/bib_ref] , based on exact alignment of k-mers to the database of known species, often outperforms MetaPhyler and other methods both in terms of speed and discovery of true positives, as indicated by our tests.
MetaCRAM employs Kraken as a default tool in the pipeline. Kraken produces an output report which is automatically processed by MetaCRAM. Part of the report contains information about species present in the sample, as well as their abundance. We rank order the species in from the most abundant to the least abundant, where abundance is based on the number of reads identified to match a species in the database. For downstream analysis, MetaCRAM selects the "most relevant" species and uses their genomes as references. The default definition of "most relevant" is the top 75 species, but one has the option to choose a threshold for the abundance value or for the number of references used. As an illustration, [fig_ref] Table 1: Analysis of the influence of different threshold values on reference genome selection... [/fig_ref] lists the results of an analysis of the impact of different thresholds on the processing time and the compression ratio.
## Alignment and assembly
After a group of reference genomes is carefully chosen based on the Kraken software output, alignment of reads to the reference genomes is performed. This task is accomplished by using Bowtie2, a standard software tool for ultra-fast alignment of short reads to long genomes. The alignment information is stored in a SAM (Sequence Alignment/Map) file format and subsequently used for compression via reference-based algorithms.
Due to the fact that many species in a metagenome sample have never been sequenced before, some reads will not be aligned to any of the references with high alignment scores, and we collectively refer to them as unaligned reads hereafter. In order to discover reference genomes for unaligned reads, we assemble the unaligned reads in a relatively efficient, although often time consuming manner using a metagenomic assembler. Our metagenomic assembler of choice is IDBA-UD [bib_ref] Idba-ud: a de novo assembler for single-cell and metagenomic sequencing data with..., Peng [/bib_ref] , given that in our tests it produced the largest number of contigs leading to new reference identification. Alternatives to IDBA-UD include the Ray Meta software [bib_ref] Ray meta: scalable de novo metagenome assembly and profiling, Boisvert [/bib_ref].
When the reads have high sequencing depth and large overlaps, the contigs produced by the assembler may be queried using BLAST to identify the organisms they most likely originated from. The user may choose to BLAST only the top n longest contigs, where n is a user specified number, but in our analysis we use all contigs (which is also the default setting). Subsequently, we align the unaligned reads to the newly found references.
In some rare cases, the assembler may fail depending on the number of species in the metasample and the sequencing depth, in which case one may want to skip the assembly step and compress the unaligned reads in a reference-free manner. For reference-free compression, the software of choice in MetaCRAM is MFCompress [bib_ref] Mfcompress: a compression tool for fasta and multi-fasta data, Pinho [/bib_ref]. As long as the assembler is successful, one can reduce the volume of unaligned reads by iterating the process of assembly, BLAST, and alignment as illustrated at the top right hand of . All our demonstrations and results are based on two iterations of the described algorithmic loop.
## Distribution of read starting positions
We empirically studied the distribution of integers representing the read positions, variation positions, and pairedend offsets in order to choose the most suitable compression method. As an example, the distribution of the starting positions for the reads that aligned to JH603150 (genome of Klebsiella oxytoca) in the dataset SRR359032 is shown in [fig_ref] Figure 4: Integer Distribution [/fig_ref]. This distribution was truncated after achieving a 90 % coverage of the data (i.e., after only 10 % of the read start positions exceeded the depicted maximum length). The empirical distribution is shown in yellow, while a fitted power law distributions is plotted and determined according to [bib_ref] Extended golomb code for integer representation, Somasundaram [/bib_ref] , with P i = 2 − log m i 1 2i(m−1) , where i is the integer to be encoded, and m is the divisor in the extended Golomb code. The parameter choose shown is m = 3 and 4. The negative binomial distribution is fitted using Maximum Likelihood Estimation (MLE), while the Geometric distribution is fitted by two different means: using MLE and ezfit, a MATLAB script which performs an unconstrained nonlinear minimization of the sum of squared residuals with respect to various parameters.
For single reference alignment methods, it was reported that the best fit for the empirical distribution is a geometric distribution or a negative binomial distribution [bib_ref] Genomic mapping by fingerprinting random clones: a mathematical analysis, Lander [/bib_ref]. However, due to sequencing errors and non-uniform distributions of hydrogen bond breakage, the empirical data often deviates from geometric or negative binomial distributions [bib_ref] Substantial biases in ultra-short read data sets from high-throughput dna sequencing, Dohm [/bib_ref]. In addition, for metagenomic samples, there exist multiple references which may have good alignments with reads that did not originally correspond to the genomic sample of the reference. This creates additional changes in the read starting position with respect to the geometric distribution. Moreover, one has to encode not only the read positions but also the variation positions and paired-end offsets, making it difficult to claim any one of the fitted distributions is better than others. This observation is supported by [fig_ref] Figure 4: Integer Distribution [/fig_ref]. Since there is no known efficient optimal encoding method for a set of integers with negative binomial distributions, and Golomb and extended Golomb encoding are optimal for geometric distributions and power law distributions, respectively, we use these two methods with m = 3. The parameter m is chosen based on extensive experiments, although the user has the freedom to adjust and modify its value.
As the number of unaligned reads that remains after a few iterations of MetaCRAM is relatively small, these reads were compressed using a reference-free tool such as MFCompress [bib_ref] Mfcompress: a compression tool for fasta and multi-fasta data, Pinho [/bib_ref] , which is based on finite-context models. Furthermore, the SAM files produced after running Bowtie2 are converted to the sorted and indexed binary format of a BAM file using SAMtools [bib_ref] The sequence alignment/map format and samtools, Li [/bib_ref]. Each BAM file is compressed via reference-based compression against its representative to a standard CRAM format. We tested three different modes of the CRAM toolkit: Huffman, Golomb, and Extended Golomb encoding, all of which are described in the next section. Note that the Extended Golomb encoding method is our new addition to the classical CRAM method, as it appears to offer good compromises between compression and decompression speed and compression ratios.
Intrinsically, SAM files contain quality values and unique read IDs for each read, which inevitably account for a large file size: quality values are characters of length as long as the sequence, and read IDs often repeat the name of the dataset. By default, MetaCRAM preserves all quality values and read IDs as designed in CRAM.
## Compression
Compression in the reference-based mode is accomplished by compressing the starting points of references with respect to the reference genomes and the base differences between the reads and references. As both the starting points and bases belong to a finite integer alphabet, we used three different integer compression methods, briefly described below.
Huffman coding is a prefix-free variable length compression method for known distributions [bib_ref] A method for the construction of minimum redundancy codes, Huffman [/bib_ref] which is information-theoretically optimal. The idea is to encode more frequent symbols with fewer bits than non-frequent ones. For example, given an alphabet A = (a, b, c, d, e) and the corresponding distribution P = (0.25, 0.25, 0.2, 0.15, 0.15), building a Huffman tree results in the codebook C = (00, 10, 11, 010, 011). Decoding relies on the Huffman tree constructed during encoding which is stored in an efficient manner, usually ordered according to the frequency of the symbol. Due to the prefix-free property, Huffman coding is uniquely decodable coding and does not require any special marker between words. Two drawbacks of Huffman coding that make it a costly solution for genomic compression are its storage complexity, since we need to record large tree structures for big alphabet size which arise when encoding positions in long sequences and the need to know the underlying distribution a priori. Adaptive Huffman coding mitigates the second problem, at the cost of increased computational complexity associated with constructing multiple encoding trees [bib_ref] Algorithms for adaptive huffman codes, Cormack [/bib_ref]. In order to alleviate computational challenges, we implemented so called canonical Huffman encoding, which bypasses the problem of storing a large code tree by sequentially encoding lengths of the codes [bib_ref] Managing Gigabytes: Compressing and Indexing Documents and Images: Morgan Kaufmann, Witten [/bib_ref].
Golomb codes are optimal prefix-free codes for countably infinite lists of non-negative integers following a geometric distribution [bib_ref] Run-length encodings, Golomb [/bib_ref]. In Golomb coding, one encodes an integer n in two parts, using its quotient q and remainder r with respect to the divisor m. The quotient is encoded in unary, while the remainder is encoded via truncated binary encoding. Given a list of integers following a geometric distribution with known mean μ, the dividend m can be optimized so as to reduce code length. In, the optimal value of m was derived for m = 2 k , for any integer k. The encoding is known as the Golomb-Rice procedure, and it proceeds as follows: first, we let
[formula] k * = max 0, 1 + log 2 log(φ−1) log μ μ+1 , where φ = ( √ 5+1) 2 . [/formula]
Unary coding represents an integer i by i ones followed by a single zero. For example, the integer i = 4 in unary reads as 11110. Truncated binary encoding is a prefix-free code for an alphabet of size m, which is more efficient than standard binary encoding. Because the remainder r can only take values in {0,1,. . . , m-1}, according to truncated binary encoding, we assign to the first 2 k+1 − m symbols codewords of fixed length k. The remaining symbols are encoded via codewords of length k + 1, where k = log 2 (m) . For instance, given n = 7 and m = 3, we have 7 = 2 × 3 + 1, implying q = 2 and r = 1. Encoding 2 in unary gives 110 and 1 in truncated binary reads as 10. Hence, the codeword used to encode the initial integer is the concatenation of the two representations, namely 11010. Decoding of Golomb encoded codewords is also decoupled into decoding of the quotient and the remainder. Given a codeword, the number of ones before the first zero determines the quotient q, while the remaining k or k + 1 bits, represents the remainder r according to truncated binary decoding for an alphabet of size m. The integer n is obtained as n = q × m + r.
Golomb encoding has one advantages over Huffman coding in so far that it is computationally efficient (as it only requires division operations). One does not need to the distribution a priori, although there are clearly no guarantees that Golomb coding for an unknown distribution will be even near-optimal: Golomb encoding is optimal only for integers following a geometric distribution.
An extension of Golomb encoding, termed extended Golomb [bib_ref] Extended golomb code for integer representation, Somasundaram [/bib_ref] coding, is an iterative method for encoding non-negative integers following a power law distribution. One divides an integer n by m until the quotient becomes 0, and then encodes the number of iterations M in unary, and an array of remainders r according to an encoding table. This method has an advantage over Golomb coding when encoding large integers, such is the case for read position compression. As an example, consider the integer n = 1000: with m = 2, Golomb coding would produce q = 500 and r = 0, and unary encoding of 500 requires 501 bits. With extended Golomb coding, the number of iterations equals M = 9 and encoding requires only 10 bits. As an illustration, let us encode n = 7 given m = 3. In the first iteration, 7 = 2 × 3 + 1, so r 1 = 1 is encoded as 10, and q 1 = 2. Since the quotient is not 0, we iterate the process: 2 = 0 × 3 + 2 implies r 2 = 2, which is encoded as 1, and q 2 = 0. Because the quotient is at this step 0, we encode M = 2 as 110 and r = r 2 r 1 = 110, and our codeword is 110110.
The decoding of extended Golomb code is also performed in M iterations. Since we have a remainder stored at each iteration and the last quotient equals q M = 0, it is possible to reconstruct the original integer. Similar to Golomb coding, extended Golomb encoding is computationally efficient, but optimal only for integers with power law distribution.
There are various other methods for integer encoding, such as Elias Gamma and Delta Encoding, which are not pursued in this paper due to the fact that they do not appear to offer good performance for the empirical distributions observed in our read position encoding experiments.
## Products
The compressed unaligned reads, CRAM files, list of reference genomes (optional), alignment rate (optional), contig files (optional) are all packaged into an archive. The resulting archive can be stored in a distributed manner and when desired, the reads can be losslessly reconstructed via the CRAM toolkit. Additional file 3 contains software instructions, and detailed descriptions of created files and folders by MetaCRAM processing are available in Additional file 4.
## Decompression
Lossless reconstruction of the reads from the compressed archive is done in two steps. For those reads with known references in CRAM format, decompression is performed with an appropriate integer decompression algorithm. When the files are converted back into the SAM format, we retrieve only the two necessary fields for FASTA format, i.e., the read IDs and the sequences printed in separate lines. Unaligned reads are decompressed separately, through the decoding methods used in MFCompress.
## Post-processing
The two parts of reads are now combined into one file, and they are sorted by the read IDs in an ascending order. If the reads were paired-end, they are separated into two files according to the mate "flag" assigned in the processing step.
## Effects of parallelization
One key innovation in the implementation of MetaCRAM is parallelization of the process, which was inspired by parallel single genome assembly used in TIGER [bib_ref] Tiger: tiled iterative genome assembler, Wu [/bib_ref]. Given that metagenomic assembly is computationally highly demanding, and in order to fully utilize the computing power of a standard desktop, MetaCRAM performs meta assembly of unaligned reads and compression of aligned reads in parallel. As shown in [fig_ref] Table 6: Processing time improvements for two rounds of MetaCRAM on the SRR359032 dataset [/fig_ref] , parallelization improves real, user, and system time by 23-40 %.
[fig] For: short hand notation, we used"MCH" = MetaCRAM-Huffman, "MCG" = MetaCRAM-Golomb, "MCEG" = MetaCRAM-extended Golomb, "MFComp" = MFCompress. MCH1 is the default option of MetaCRAM with Huffman encoding, and MCH2 is a version of MetaCRAM in which we removed the redundancy in both quality scores and the read IDs. "Align. %" refers to the total alignment rates from the first and second iteration. Minimum compressed file size achievable by the methods are written in bold case letters. Minimum compressed file size achievable by the methods are written in bold case letters [/fig]
[fig] Figure 2: Compression ratio. The compression ratios for all six software suites, indicating the compression ratio [/fig]
[fig] Figure 3: Average Runtime of Each Stage of MetaCRAM. Detailed distribution of the average runtimes of MetaCRAM for all five datasets tested. We used "_1" to indicate the processes executed in the first round, and "_2" to denote the processes executed in the second round [/fig]
[fig] Figure 4: Integer Distribution. Distribution fitting of integers to be encoded, truncated at 90 % of the integer data [/fig]
[table] Table 1: Analysis of the influence of different threshold values on reference genome selection after taxonomy identification and compression ratios [/table]
[table] Table 2: Comparison of compression ratios of six software suites [/table]
[table] Table 3: Comparison of processing (compression) times of six software suites. Times are recorded row by row denoting real, user, and system time in order [/table]
[table] Table 4: Comparison of compressed file sizes of MetaCRAM-Huffman using 2 rounds and 1 round [/table]
[table] Table 5: Comparison of retrieval (decompression) times of six software suites. Times are recorded row by row denoting real, user, and system time in order [/table]
[table] Table 6: Processing time improvements for two rounds of MetaCRAM on the SRR359032 dataset (5.4 GB, without removing redundancy in description lines) resulting from parallelization of assembly and compression [/table]
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Visceral Adipose Tissue E2F1-miRNA206/210 Pathway Associates with Type 2 Diabetes in Humans with Extreme Obesity
Citation: Maixner, N.; Haim, Y.; Blüher, M.; Chalifa-Caspi, V.; Veksler-Lublinsky, I.; Makarenkov, N.; Yoel, U.; Bashan, N.; Liberty, I.F.; Kukeev, I.; et al. Visceral Adipose Tissue E2F1-miRNA206/210 Pathway Associates with Type 2 Diabetes in Humans with Extreme Obesity. Cells 2022, 11, 3046. https://doi.
# Introduction
Obesity, the excessive fat accumulation that impinges on health, is a highly heterogeneous disease, presenting itself differently in patients with similar degrees of adiposity as reflected by body mass index (BMI) [bib_ref] Obesity phenotypes and their paradoxical association with cardiovascular diseases, Vecchié [/bib_ref] [bib_ref] Insulin-sensitive obesity, Klöting [/bib_ref]. It is now well-established that this clinical heterogeneity is paralleled to, and may be attributed to, the different degrees of metabolic dysfunction that adipose tissue develops in response to excess fat accumulation [bib_ref] Insulin-sensitive obesity, Klöting [/bib_ref] [bib_ref] Metabolically healthy obesity: Epidemiology, mechanisms, and clinical implications, Stefan [/bib_ref] [bib_ref] Identification and Characterization of Metabolically Benign Obesity in Humans, Stefan [/bib_ref] [bib_ref] The distinction of metabolically 'healthy' from 'unhealthy' obese individuals, Blüher [/bib_ref]. The molecular basis of adipose tissue dysfunction includes pathways involved in the initiation and maintenance of chronic low-grade inflammation [bib_ref] The pathogenesis of obesity-associated adipose tissue inflammation, Engin [/bib_ref] [bib_ref] Regulation of adipose tissue inflammtion by interleukin, Han [/bib_ref] , molecular stress-pathway activation [bib_ref] Altered Autophagy in Human Adipose Tissues in Obesity, Kovsan [/bib_ref] [bib_ref] Mitogen-activated protein kinases, inhibitory-κB kinase, and insulin signaling in human omental versus..., Bashan [/bib_ref] [bib_ref] ASK1 (MAP3K5) is transcriptionally upregulated by E2F1 in adipose tissue in obesity,..., Haim [/bib_ref] [bib_ref] Elevated autophagy gene expression in adipose tissue of obese humans: A potential..., Haim [/bib_ref] [bib_ref] Activated Ask1-MKK4-p38MAPK/JNK Stress Signaling Pathway in Human Omental Fat Tissue May Link..., Blüher [/bib_ref] , and altered gene expression governed by transcription factors, the miRNA landscape, and epigenetic changes [bib_ref] MicroRNA regulatory networks in human adipose tissue and obesity, Arner [/bib_ref] [bib_ref] Adipose Tissue MicroRNAs as Regulators of CCL2 Production in Human Obesity, Arner [/bib_ref] [bib_ref] Additive Effects of MicroRNAs and Transcription Factors on CCL2 Production in Human..., Kulyté [/bib_ref] [bib_ref] Meta-analysis of gene expression data in adipose tissue reveals new obesity associated..., Goutzelas [/bib_ref]. The motivation for exploring these molecular alterations of adipose tissue in a metabolically dysfunctional obesity phenotype is in the hope of identifying both markers and drivers that would inspire better obesity subtype classification and eventual development of more personalized therapeutics.
Over recent years, E2F1 has been identified as a molecular hub regulating stress and inflammatory pathways within and around adipocytes in both human and murine adipose tissue [bib_ref] ASK1 (MAP3K5) is transcriptionally upregulated by E2F1 in adipose tissue in obesity,..., Haim [/bib_ref] [bib_ref] Elevated autophagy gene expression in adipose tissue of obese humans: A potential..., Haim [/bib_ref] [bib_ref] A TRAIL-TL1A Paracrine Network Involving Adipocytes, Macrophages, and Lymphocytes Induces Adipose Tissue..., Maixner [/bib_ref]. In obesity, E2F1 is elevated in the adipocyte cell fraction of visceral adipose tissue (VAT) [bib_ref] Elevated autophagy gene expression in adipose tissue of obese humans: A potential..., Haim [/bib_ref]. Traditionally studied as a cell-cycle progression regulator, in the largely quiescent adipocytes, this transcription factor appears to play different roles, driving the expression and activation of stress-activated MAPK cascades [bib_ref] ASK1 (MAP3K5) is transcriptionally upregulated by E2F1 in adipose tissue in obesity,..., Haim [/bib_ref] and of autophagy genes [bib_ref] Elevated autophagy gene expression in adipose tissue of obese humans: A potential..., Haim [/bib_ref]. In pre-adipocytes, E2F1 acts as an adipogenic stimulator [bib_ref] Impaired pancreatic growth, β cell mass, and β cell function in E2F1..., Fajas [/bib_ref] and negatively regulates energy expenditure and mitochondrial activity via the transcriptional regulation of mitochondrial genes [bib_ref] Acute loss of transcription factor E2F1 induces mitochondrial biogenesis in Hela cells, Goto [/bib_ref] [bib_ref] E2F transcription factor-1 regulates oxidative metabolism, Blanchet [/bib_ref]. Additionally, increased E2F1 expression in human VAT is associated with altered TNF superfamily gene expression, which maintains an inflammatory paracrine loop between adipocytes and immune cells in the tissue [bib_ref] A TRAIL-TL1A Paracrine Network Involving Adipocytes, Macrophages, and Lymphocytes Induces Adipose Tissue..., Maixner [/bib_ref]. At the whole-body level, E2F1 mRNA expression in human VAT correlates with insulin resistance, circulating IL-6, leptin, and FFA levels [bib_ref] Elevated autophagy gene expression in adipose tissue of obese humans: A potential..., Haim [/bib_ref]. Taken together, E2F1 is an emerging contributor to the endocrine-metabolic dysfunction of adipocytes and adipose tissue.
In multiple cancers (an area in which E2F1 was most studied), E2F1 expression levels were noted to be tightly connected to the abundance of specific miRNAs. In fact, a complex co-regulatory network of E2F1 and microRNAs was described and termed the "E2F1-microRNA-cancer progression network". This network consists of E2F1 as a central hub of multiple bi-directional regulatory loops, in which E2F1 governs the transcription of specific miRNAs, and these, in turn, feed back to affect its expression, either directly or via another mediating transcription factor. In colon-cancer cells, for instance, miRNA cluster members miR-17-92, 106a-363, and 106b-25 are all transcriptionally induced by E2F1 [bib_ref] An E2F/miR-20a Autoregulatory Feedback Loop, Sylvestre [/bib_ref] [bib_ref] Direct Regulation of an Oncogenic Micro-RNA Cluster by E2F Transcription Factors, Woods [/bib_ref] [bib_ref] Emerging role of miR-106b-25/miR-17-92 clusters in the control of transforming growth factor..., Petrocca [/bib_ref] [bib_ref] c-Myc-regulated microRNAs modulate E2F1 expression, O'donnell [/bib_ref] [bib_ref] The c-Myc-Regulated MicroRNA-17∼92 (miR-17∼92) and miR-106a∼363 Clusters Target hCYP19A1 and hGCM1 To..., Kumar [/bib_ref]. They are also up-regulated by MYC, which is itself induced by E2F1 [bib_ref] The c-Myc-Regulated MicroRNA-17∼92 (miR-17∼92) and miR-106a∼363 Clusters Target hCYP19A1 and hGCM1 To..., Kumar [/bib_ref]. In turn, the three miRNA clusters inhibit E2F1's translation directly, creating an auto-feedback regulatory loop. Another miRNA family displaying a complex interaction with E2F1 is the miRNA-449a/b/c cluster, all direct transcriptional targets of E2F1 [bib_ref] miR-449a and miR-449b are direct transcriptional targets of E2F1 and negatively regulate..., Yang [/bib_ref]. Upon their induction, miRNA-449a/b inhibit CDK2 and CDC25A (activators of Rb-phosphorylation), thereby inhibiting E2F1 release and activation [bib_ref] miR-449 regulates CDK-Rb-E2F1 through an auto-regulatory feedback circuit, Feng [/bib_ref]. Similar regulatory loops were also described between E2F1 and miRNAs-34a, -15, -16, -205, and others, thoroughly reviewed in. These molecular interactions comprise intricate control over E2F1's expression and activity in the malignant context. The existence of these interactions in other biological contexts, such as the adipose tissue in obesity, was not examined so far. This is despite an expanding body of literature implicating adipose tissue miRNAs in regulating adipose tissue and whole-body metabolic regulation [bib_ref] Contributions of microRNAs to Peripheral Insulin Sensitivity, Kim [/bib_ref].
In light of the above, we hypothesized that in human obesity adipose tissue harbors a functional E2F1-miRNA network similar to (or, alternatively, distinct from) the previously described E2F1-miRNA-cancer progression network. Up-regulated miRNAs in human VAT in obesity may act as mediators of E2F1, linking high E2F1 with its related dysmetabolic obesity phenotype.
# Methods
## Human cohorts and vat samples
Participants were recruited from two independent adipose tissue bio-banks, in Beer-Sheva, Israel and in Leipzig, Germany. As described in previous publications [bib_ref] Elevated autophagy gene expression in adipose tissue of obese humans: A potential..., Haim [/bib_ref] [bib_ref] A TRAIL-TL1A Paracrine Network Involving Adipocytes, Macrophages, and Lymphocytes Induces Adipose Tissue..., Maixner [/bib_ref] , the two bio-banks used a coordinated methodology for adipose tissue collection, samples' processing, and storage. Ethical approval of the study procedures was obtained by the ethics committees of the 2 centers: Ethics Committee of the University of Leipzig, approval no: 159-12-21052012, and Helsinki Ethics Committee of Soroka University Medical Center, approval no: 15-0348. All participants signed a written informed consent form after being explained the study's procedures. Participants (18-75 years old) were recruited, before undergoing elective abdominal surgeries (bariatric or other elective procedures). Following overnight fasting, blood samples were drawn and analyzed by the clinical biochemistry and endocrinology labs for routine parameters. Visceral adipose tissue biopsies were obtained from the greater momentum during surgery and immediately delivered to the laboratory, where they were snap-frozen using liquid nitrogen, and then processed for miRNA/mRNA/protein extraction using coordinated procedures, as in [bib_ref] Elevated autophagy gene expression in adipose tissue of obese humans: A potential..., Haim [/bib_ref].
## Cohorts assembly
Both cohorts were designed to include age sex, ±BMI -matched participants who differed in the VAT expression level of E2F1. In cohort 1 the range of E2F1 protein content in VAT was determined in samples of n = 67 donors of the Beer-Sheva bio-bank by western blotting, as previously described in [bib_ref] Elevated autophagy gene expression in adipose tissue of obese humans: A potential..., Haim [/bib_ref] , using anti-E2F1-mAb (GenTex, GTX-70154, Irvine, CA, USA). High-E2F1 expression was defined as the expression level in the upper 40% (i.e., 2 quintiles) of the E2F1 expression range, and low-E2F1 expression in the lower 40% (2 quintiles).
Donors in the middle quintile of VAT-E2F1 expression level were excluded to minimize misclassification bias. From the patients with high and low VAT-E2F1 expression range, we matched triplets of patients for age, sex, and BMI. We assembled triplets, each including a patient without obesity and with low E2F1, a patient with obesity and low E2F1, and a participant with obesity and high E2F1. Of note, samples of the 8 pairs from patients with obesity were already used and reported in [bib_ref] A TRAIL-TL1A Paracrine Network Involving Adipocytes, Macrophages, and Lymphocytes Induces Adipose Tissue..., Maixner [/bib_ref] for other analyses (RNA-seq). The clinical characteristics of Cohort 1 participants are presented in [fig_ref] Table 1: Clinical characteristics of participants in cohort 1 [/fig_ref]. Cohort 2 consisted of patients with extreme obesity, without or with T2DM. The range of E2F1 mRNA levels was determined by quantitative rtPCR, as follows: out of a previously described cohort of 438 adipose tissue donors [bib_ref] Elevated autophagy gene expression in adipose tissue of obese humans: A potential..., Haim [/bib_ref] , we selected ten age, sex, and BMI-matched pairs from the highest and lowest E2F1 mRNA expression quintile. All patients were with extreme obesity (i.e., BMI ≥ 45 kg/m 2 ). RNA from VAT samples was extracted using RNeasy Lipid tissue Mini Kit (74804, Qiagen, Hilden, Germany). Quantity and integrity of RNA were monitored with NanoVue plus Spectrophotometer (GE Healthcare, Freiburg, Germany). An amount of 1 µg of total RNA was reverse-transcribed with a high-capacity cDNA reverse transcriptase kit (4374966, Life Technologies, Darmstadt, Germany). cDNA was then processed for TaqMan probe-based quantitative real-time polymerase chain reaction (qPCR) using the QuantStudio 6 Flex Real-Time PCR System (Life Technologies, Darmstadt, Germany). The expression of E2F1 was calculated by the standard curve method and normalized to the expression of hypoxanthine-guanine phosphoribosyltransferase 1 (HPRT1) as a housekeeping gene. The clinical characteristics of Cohort 2 participants are presented in [fig_ref] Table 2: Clinical characteristics of participants in cohort 2 [/fig_ref].
## Mirna extraction and quantification and nanostring©
Adipose tissue samples were lysed using the OMNI TH tissue homogenizer (OMNI international) and QIAzol lysis reagent (Qiagen, 79306). Total RNA was extracted using miRNeasy mini kit (Qiagen, 217004). The extracted amount was quantified using nanodrop©. An amount of 2 µg of total RNA per sample was reverse-transcribed with Taq-Man Advanced miRNA cDNA synthesis kit (Life Technologies, A28007). TaqMan system (Life Technologies, 4369016) including specific TaqMan assays for miR-206 (477968_mir), miR-210-5p (478765_mir), and miR-let-7b (478576_mir), (Life Technologies) was used for real-time PCR amplification. Relative miRNA expression was obtained after normalization to an endogenous control (miR-let-7b), using the formula 2 −∆∆Ct . Unbiased miRNA screening in adipose tissue was performed using the Nanostring Ncounter© technology, with the help of Agentek 2019©, Yakum, Israel. In the Nanostring run, the samples were randomly divided among 2 chips. miRNA data were analyzed in Partek Genomics Suite. Only miRNAs designated as "Endogenous1" were used. Raw signals were transformed by log2 and submitted to quantile normalization. Principal Component Analysis (PCA) and sources of variation analysis showed a clear batch effect caused by the chip and a minor batch effect by the match group. To identify differentially expressed miRNAs, a 3-way ANOVA was performed per each miRNA, with the patient group as a fixed effect and two random (batch) effects: chip and match group. The ANOVA model included the following contrasts: obese E2F1-high vs. obese E2F1-low, obese E2F1-low vs. lean E2F1-low, and obese E2F1-high vs. lean E2F1-low. The p-values were adjusted for multiple testing using the FDR method; however, no miRNA passed the p = 0.05 cutoff in any of Cells 2022, 11, 3046 5 of 18 the contrasts. Therefore, given that nanostring© analysis provided merely the baseline for further molecular experimental approaches, we enabled permissive statistical cutoffs. In all subsequent analyses, nominal p-values were used with a 0.05 cutoff, and a fold change cutoff of 1.25. The fold of change values was written in a linear scale (minus sign indicating down-regulation).
## Cellular studies
Human embryonic kidney (HEK)-293 cells (ATCC, Manassas, VA, USA) were grown in DMEM containing 4.5 mM glucose, 10% FBS, 2 mM L-glutamin, and 100 U/mL penicillinstreptomycin. The medium was changed every other day. Cells were grown to 80% confluence, then transfected with siRNA/plasmid and subjected to total RNA extraction as detailed below. For miRNA quantification following manipulations, frozen cells were scraped on ice with QIAzol lysis reagent (Qiagen, 79306) and the total RNA was extracted and miRNA was quantified using similar procedures as described for adipose tissue samples. To overexpress or knockdown E2F1, cells were seeded in 6 well plates in 1 mL/well of DMEM 10% FCS and grown to 80% confluence. To overexpress E2F1, the medium was changed to fresh medium, 1 mL/well, and cells were transfected with 200 ng/well of pCMV-E2F1 or empty plasmid, using jetPRIME reagent (Poly-plus transfection Inc., New York, NY, USA), according to the manufacturer's instructions. To knock-down E2F1, small interfering RNA(siRNA) directed against E2F1, or a non-specific (ns) control (negative control) siRNAs, ON-TARGETplus smart pools were obtained from Dharmacon (Thermo Fisher Scientific Inc., Waltham, MA, USA). Cells were transfected with 4 ng/well siRNA using jetPRIME reagent. After transfection with either plasmid or siRNA, cells were incubated for 4 h, washed, and incubated for an additional 44 h with fresh media. After a total of 48 h, cells were snap-frozen and processed for miRNA determination as described above.
## E2f1 chip-seq data collection from public databases
The information for the E2F1 binding site was obtained from Chip-Atlas [bib_ref] ChIP-Atlas 2021 update: A data-mining suite for exploring epigenomic landscapes by fully..., Zou [/bib_ref] -an integrative database covering all public Chip-seq data submitted to the NCBI SRA. The following data sets were used: Mammary gland (GSM1526876), Mesenchymal Stem Cells (GSM2046834), Hela (GSM558469), SK-MEL-147 (GSM1665998), U-266 (GSM2132555), RAJI (GSM1976292), MCF-7 (GSM699985), MDA-MB-231 (GSM2501567), and K562 cells (GSM2827553). IGV browser version 2.14.0 was used for visualization [bib_ref] Integrative genomics viewer, Robinson [/bib_ref].
## Statistical analyses
The T-test/Mann-Whitney test for a 2-group comparison and Pearson's correlation test for correlation analysis were conducted using Graphpad-Prism 9.1.0. To enable the combined analyses of the two cohorts (cohort 1-Beer-Sheva, Israel; cohort 2-Leipzig, Germany) despite operator-dependent differences between cohorts, data were presented as fold-change of the mean. Quantitative variables (expression levels, FPG, FPI) were handled as continuous variables. Missing values were excluded from the analysis and not extrapolated.
# Results
## E2f1-mirna associations in vat are distinct from the e2f1-mirna cancer-associated co-regulation network
To search for E2F1-associated VAT miRNAs, we utilized VAT samples from an assembled cohort consisting of n = 8 triplets (i.e., in total n = 24 participants, Cohort 1). To avoid any potential bias by background parameters, patients were matched from a pre-screened cohort of n = 67 for VAT-E2F1 protein level, age, sex, and BMI as follows: each triplet consisted of a patient with obesity and high VAT-E2F1 expression; an age, sex and BMI-matched participant with obesity and low VAT-E2F1 expression; and an age and sex-matched patient without obesity and with low VAT-E2F1 expression. The two matched groups of patients with obesity were previously reported [bib_ref] A TRAIL-TL1A Paracrine Network Involving Adipocytes, Macrophages, and Lymphocytes Induces Adipose Tissue..., Maixner [/bib_ref] , and more details are described in Methods (Section 2.2. Cohorts Assembly). Patients' characteristics and triplet matching with the E2F1 expression, age, and BMI of Cohort 1 are shown in [fig_ref] Table 1: Clinical characteristics of participants in cohort 1 [/fig_ref] and [fig_ref] Figure 1: Figure 1 [/fig_ref] -C, respectively.
## Co-regulation network
To search for E2F1-associated VAT miRNAs, we utilized VAT samples from an assembled cohort consisting of n = 8 triplets (i.e., in total n = 24 participants, Cohort 1). To avoid any potential bias by background parameters, patients were matched from a prescreened cohort of n = 67 for VAT-E2F1 protein level, age, sex, and BMI as follows: each triplet consisted of a patient with obesity and high VAT-E2F1 expression; an age, sex and BMI-matched participant with obesity and low VAT-E2F1 expression; and an age and sexmatched patient without obesity and with low VAT-E2F1 expression. The two matched groups of patients with obesity were previously reported [bib_ref] A TRAIL-TL1A Paracrine Network Involving Adipocytes, Macrophages, and Lymphocytes Induces Adipose Tissue..., Maixner [/bib_ref] , and more details are described in Methods (Section 2.2. Cohorts Assembly). Patients' characteristics and triplet matching with the E2F1 expression, age, and BMI of Cohort 1 are shown in [fig_ref] Table 1: Clinical characteristics of participants in cohort 1 [/fig_ref] and [fig_ref] Figure 1: Figure 1 [/fig_ref] -C, respectively.
## Figure 1.
Two cohorts of matched patients with high-vs. low-E2F1 levels in VAT. Cohort 1: Human adipose tissue E2F1 protein expression range was determined on a pre-screened cohort of n = 67 persons, and high and low expression were those in the upper and lower two quintiles (40%), respectively. Based on this definition, cohort 1 (Beer-Sheva, Israel), consisted of age, sex, and (when relevant) BMI-matched triplets, each consisting of patients "without obesity and with low-E2F1 expression", "with obesity and low-E2F1 expression" and "with obesity and high-E2F1 expression". The matching of eight triplets of patients (i.e, n = 8 × 3 = 24) for E2F1 expression (A), BMI (B), and age (C) is shown. Cohort 2 (Leipzig, Germany) consisted of 10 similarly matched patients, with the following differences: E2F1 expression definition and matching; (D) was based on mRNA expression, and the cohort included pairs only of patients with extreme obesity (BMI ≥ 45 kg/m 2 ) also matched for BMI (E) and age (F). Different colors represent each matched couple/triplet. Dots represent individual patients. *** = Pv < 0.001, **** = Pv < 0.0001, ns = non-significant. Two cohorts of matched patients with high-vs. low-E2F1 levels in VAT. Cohort 1: Human adipose tissue E2F1 protein expression range was determined on a pre-screened cohort of n = 67 persons, and high and low expression were those in the upper and lower two quintiles (40%), respectively. Based on this definition, cohort 1 (Beer-Sheva, Israel), consisted of age, sex, and (when relevant) BMI-matched triplets, each consisting of patients "without obesity and with low-E2F1 expression", "with obesity and low-E2F1 expression" and "with obesity and high-E2F1 expression". The matching of eight triplets of patients (i.e., n = 8 × 3 = 24) for E2F1 expression (A), BMI (B), and age (C) is shown. Cohort 2 (Leipzig, Germany) consisted of 10 similarly matched patients, with the following differences: E2F1 expression definition and matching; (D) was based on mRNA expression, and the cohort included pairs only of patients with extreme obesity (BMI ≥ 45 kg/m 2 ) also matched for BMI (E) and age (F). Different colors represent each matched couple/triplet. Dots represent individual patients. *** = Pv < 0.001, **** = Pv < 0.0001, ns = non-significant. miRNAs were isolated from VAT of all (i.e., 3 × 8) 24 participants, and identified using the nCounter ® miRNA expression panel. Nanostring© technology provides a hybridization-based, amplification-free direct count of miRNA copy numbers. Overall, 798 miRNAs were identified, out of which 47 (5.7%) were differentially expressed (DE), with a fold change (FC) > 1.25 or <−1.25, p < 0.05, in at least one inter-group comparison . Notably, a heatmap of miRNA expression profiles uncovers miRNAs primarily altered by obesity status (clusters 2 and 5, , being down-and up-regulated, respectively, in patients with obesity compared to those without, regardless of VAT E2F1 expression level. In contrast, other clusters of altered miRNAs exhibited differences primarily associated with E2F1 expression (clusters 1, 3, and 4, . We focused on the seven overlapping VAT miRNAs between the two high/low E2F1 group comparisons, to reflect those that mainly corresponded to differences in E2F1 expression irrespective of obesity (represented by the indicated area in the Venn diagram, . These seven miRNAs included miR-210-5p, miR-206, and its cluster-mate miRNA-1-3p, which were significantly up-regulated in high-versus low-VAT E2F1 samples, while miRNA-375, miRNA-498, miRNA-504-3p, and miRNA-323a-3p were downregulated.
bridization-based, amplification-free direct count of miRNA copy numbers. Overall, 798 miRNAs were identified, out of which 47 (5.7%) were differentially expressed (DE), with a fold change (FC) > 1.25 or <−1.25, p < 0.05, in at least one inter-group comparison . Notably, a heatmap of miRNA expression profiles uncovers miRNAs primarily altered by obesity status (clusters 2 and 5, , being down-and up-regulated, respectively, in patients with obesity compared to those without, regardless of VAT E2F1 expression level. In contrast, other clusters of altered miRNAs exhibited differences primarily associated with E2F1 expression (clusters 1,3, and 4, . We focused on the seven overlapping VAT miRNAs between the two high/low E2F1 group comparisons, to reflect those that mainly corresponded to differences in E2F1 expression irrespective of obesity (represented by the indicated area in the Venn diagram, . These seven miRNAs included miR-210-5p, miR-206, and its cluster-mate miRNA-1-3p, which were significantly up-regulated in high-versus low-VAT E2F1 samples, while miRNA-375, miRNA-498, miRNA-504-3p, and miRNA-323a-3p were downregulated.
## Figure 2.
Differentially expressed miRNAs between three sub-groups of cohort 1. VAT samples of cohort 1 patients underwent miRNA extraction and quantification using Nanostring Ncounter© technology. Differentially expressed (DE) miRNAs (by at least one inter-group comparison) were hierarchically clustered and divided by trends of up/down-regulation between the three cohort groups (A). miRNAs differentially expressed by at least ±1.25 with Pv ≤ 0.05 were inflicted on a Venn diagram intersecting the results of three inter-group comparisons (B). Highlighted (dashed blue line) are miRNAs commonly DE in the two E2F1-low vs. E2F1-high comparisons. Legend: green and red fonts represent downregulated and up-regulated miRNAs, respectively. Font size is inversely proportional to Pv of differential expression for each miRNA (larger font represents lower Pv and vice versa).
Overall, members of the cancer progression E2F1-miRNA network (miRNA-17-5p and 106a/b clusters, miRNA-15a/b, miRNA-16, miRNA-34a, miRNA-205, and miRNAs-449a/b/c, [fig_ref] Figure 3: Members of the cancer-associated miRNA-E2F1 co-regulatory network are not altered in association... [/fig_ref] were absent from the identified set of VAT miRNAs altered according to E2F1 expression. (Of note, miRNA-449b-5p was elevated in high VAT-E2F1 with obe- . Differentially expressed miRNAs between three sub-groups of cohort 1. VAT samples of cohort 1 patients underwent miRNA extraction and quantification using Nanostring Ncounter© technology. Differentially expressed (DE) miRNAs (by at least one inter-group comparison) were hierarchically clustered and divided by trends of up/down-regulation between the three cohort groups (A). miRNAs differentially expressed by at least ±1.25 with Pv ≤ 0.05 were inflicted on a Venn diagram intersecting the results of three inter-group comparisons (B). Highlighted (dashed blue line) are miRNAs commonly DE in the two E2F1-low vs. E2F1-high comparisons. Legend: green and red fonts represent downregulated and up-regulated miRNAs, respectively. Font size is inversely proportional to Pv of differential expression for each miRNA (larger font represents lower Pv and vice versa).
Overall, members of the cancer progression E2F1-miRNA network (miRNA-17-5p and 106a/b clusters, miRNA-15a/b, miRNA-16, miRNA-34a, miRNA-205, and miRNAs-449a/b/c, [fig_ref] Figure 3: Members of the cancer-associated miRNA-E2F1 co-regulatory network are not altered in association... [/fig_ref] were absent from the identified set of VAT miRNAs altered according to E2F1 expression. (Of note, miRNA-449b-5p was elevated in high VAT-E2F1 with obesity as compared to low VAT-E2F1, without obesity. However, this miRNA failed to differentiate between low vs. high VAT-E2F1 among patients with obesity and therefore was not further considered). sity as compared to low VAT-E2F1, without obesity. However, this miRNA failed to differentiate between low vs. high VAT-E2F1 among patients with obesity and therefore was not further considered).
## E2f1-associated changes in mirna expression are validated in an independent patient population with extreme obesity
To validate the result described above in patients with extreme obesity and either low or high E2F1 expression, we utilized VAT samples from an independent cohort, also age, sex and BMI-matched (Cohort 2, n = 20, [fig_ref] Figure 1: Figure 1 [/fig_ref]. All participants were with extreme obesity (BMI > 45 Kg/m 2 ), older than patients in Cohort 1, and, remarkably, all those with low VAT-E2F1 were without T2DM and all those with high VAT-E2F1 were with T2DM, reflecting the metabolic impact associated with high-VAT-E2F1 expression [bib_ref] Elevated autophagy gene expression in adipose tissue of obese humans: A potential..., Haim [/bib_ref] [fig_ref] Table 2: Clinical characteristics of participants in cohort 2 [/fig_ref]. The expression of selected E2F1-associated miRNAs in VAT was assessed by real-time PCR and compared to nanostring results [fig_ref] Figure 3: Members of the cancer-associated miRNA-E2F1 co-regulatory network are not altered in association... [/fig_ref]. Consistent with cohort 1, no significant changes in expression levels of the cancer progression E2F1-miRNA network members miRNA-17-5p, miRNA-106a, miRNA-106b were seen between the groups [fig_ref] Figure 3: Members of the cancer-associated miRNA-E2F1 co-regulatory network are not altered in association... [/fig_ref]. These results suggest that in VAT of patients with extreme obesity and with T2DM associated with high VAT-E2F1 expression, up-regulation of E2F1 associates with changes in miRNA expression distinct from those known in malignant disease contexts.
We next aimed to validate E2F1-associated changes in miRNA in the VAT of patients with obesity using cohort 2. miRNA-498 and 375, down-regulated in high-E2F1 VAT in
## E2f1-associated changes in mirna expression are validated in an independent patient population with extreme obesity
To validate the result described above in patients with extreme obesity and either low or high E2F1 expression, we utilized VAT samples from an independent cohort, also age, sex and BMI-matched (Cohort 2, n = 20, [fig_ref] Figure 1: Figure 1 [/fig_ref]. All participants were with extreme obesity (BMI > 45 kg/m 2 ), older than patients in Cohort 1, and, remarkably, all those with low VAT-E2F1 were without T2DM and all those with high VAT-E2F1 were with T2DM, reflecting the metabolic impact associated with high-VAT-E2F1 expression [bib_ref] Elevated autophagy gene expression in adipose tissue of obese humans: A potential..., Haim [/bib_ref] [fig_ref] Table 2: Clinical characteristics of participants in cohort 2 [/fig_ref]. The expression of selected E2F1-associated miRNAs in VAT was assessed by real-time PCR and compared to nanostring results [fig_ref] Figure 3: Members of the cancer-associated miRNA-E2F1 co-regulatory network are not altered in association... [/fig_ref]. Consistent with cohort 1, no significant changes in expression levels of the cancer progression E2F1-miRNA network members miRNA-17-5p, miRNA-106a, miRNA-106b were seen between the groups [fig_ref] Figure 3: Members of the cancer-associated miRNA-E2F1 co-regulatory network are not altered in association... [/fig_ref]. These results suggest that in VAT of patients with extreme obesity and with T2DM associated with high VAT-E2F1 expression, up-regulation of E2F1 associates with changes in miRNA expression distinct from those known in malignant disease contexts.
We next aimed to validate E2F1-associated changes in miRNA in the VAT of patients with obesity using cohort 2. miRNA-498 and 375, down-regulated in high-E2F1 VAT in cohort 1 , exhibited either no change [fig_ref] Figure 4: miRNA-206 and miRNA-210-5p intercorrelate and are candidate E2F1 targets [/fig_ref] or a non-significant trend for upregulation [fig_ref] Figure 4: miRNA-206 and miRNA-210-5p intercorrelate and are candidate E2F1 targets [/fig_ref] , respectively, in cohort 2. Yet, the expression of both miRNA-206 and 210-5p was higher in VAT samples with high E2F1 expression compared to those with low E2F1 expression [fig_ref] Figure 4: miRNA-206 and miRNA-210-5p intercorrelate and are candidate E2F1 targets [/fig_ref] ,D, respectively), corroborating the results of cohort 1. Indeed, in both cohorts, when treated as continuous variables, the expression of miRNA-206 and Cells 2022, 11, 3046 9 of 18 210-5p similarly correlated with an adjusted E2F1 expression (miRNA-206 correlated with E2F1 non-significantly, p = 0.056, in the German cohort 2) [fig_ref] Figure 4: miRNA-206 and miRNA-210-5p intercorrelate and are candidate E2F1 targets [/fig_ref]. Moreover, both cohorts exhibited a statistically significant intercorrelation between the two E2F1-associated miRNAs [fig_ref] Figure 4: miRNA-206 and miRNA-210-5p intercorrelate and are candidate E2F1 targets [/fig_ref] , possibly supporting a common up-stream regulation. The statistical significance of the results is, overall, consistent between analyses on each cohort separately, and when considered jointly as a unified cohort [fig_ref] Figure 4: miRNA-206 and miRNA-210-5p intercorrelate and are candidate E2F1 targets [/fig_ref]. We therefore focused our further investigations on these two miRNAs as putative down-stream targets of E2F1 in adipose tissue. cohort 1 , exhibited either no change [fig_ref] Figure 4: miRNA-206 and miRNA-210-5p intercorrelate and are candidate E2F1 targets [/fig_ref] or a non-significant trend for up-regulation [fig_ref] Figure 4: miRNA-206 and miRNA-210-5p intercorrelate and are candidate E2F1 targets [/fig_ref] , respectively, in cohort 2. Yet, the expression of both miRNA-206 and 210-5p was higher in VAT samples with high E2F1 expression compared to those with low E2F1 expression [fig_ref] Figure 4: miRNA-206 and miRNA-210-5p intercorrelate and are candidate E2F1 targets [/fig_ref] ,D, respectively), corroborating the results of cohort 1. Indeed, in both cohorts, when treated as continuous variables, the expression of miRNA-206 and 210-5p similarly correlated with an adjusted E2F1 expression (miRNA-206 correlated with E2F1 non-significantly, p = 0.056, in the German cohort 2) [fig_ref] Figure 4: miRNA-206 and miRNA-210-5p intercorrelate and are candidate E2F1 targets [/fig_ref]. Moreover, both cohorts exhibited a statistically significant intercorrelation between the two E2F1-associated miRNAs [fig_ref] Figure 4: miRNA-206 and miRNA-210-5p intercorrelate and are candidate E2F1 targets [/fig_ref] , possibly supporting a common up-stream regulation. The statistical significance of the results is, overall, consistent between analyses on each cohort separately, and when considered jointly as a unified cohort [fig_ref] Figure 4: miRNA-206 and miRNA-210-5p intercorrelate and are candidate E2F1 targets [/fig_ref]. We therefore focused our further investigations on these two miRNAs as putative down-stream targets of E2F1 in adipose tissue. Validation of E2F1-related DE miRNAs in human VAT using cohort 2; VAT samples of cohort 2 patients were subjected to miRNA extraction and reverse transcription as described. Expression levels of two down-regulated miRNAs in cohort 1-miR-498 (A) and miR-375 (B), and two up-regulated miR-NAs-miRNA-206 (C) and miRNA-210-5p (D) were quantified using RT-qPCR. Comparison of mean miRNA expression in patients with obesity and either low-or high-E2F1 expression was performed using Mann-Whitney non-parametric test. * = Pv < 0.05. (E-G): Inter-correlations of E2F1, miRNA-206, and miRNA-210-5p; miRNA-206 (E) and miRNA-210-5p (F) correlation with adjusted E2F1 expression in human VAT. Intercorrelation between the two miRNAs (G). Shown are data from the two cohorts-cohort 1 participants are depicted in red circles, cohort 2 in black squares. Correlation coefficients (rp) and statistical significance (Pv) for each cohort separately (Cohort 1, Beer-Sheva, Israel in red; Cohort 2, Leipzig, Germany in black) and for the joint cohort (in bold black) were analyzed using Pearson's correlation test.
## E2f1 regulation of mirna-206 and mirna-210-5p expression
To determine if E2F1 may be a direct transcriptional regulator of the two miRNAs, we first conducted a prediction analysis of putative E2F1 binding sites in the minimal promoter regions (−1000 from the transcription initiation site) of miRNA-206 and 210-5p. This DNA sequence analysis suggested a high probability for four and 10 putative binding Validation of E2F1-related DE miRNAs in human VAT using cohort 2; VAT samples of cohort 2 patients were subjected to miRNA extraction and reverse transcription as described. Expression levels of two down-regulated miRNAs in cohort 1-miR-498 (A) and miR-375 (B), and two up-regulated miRNAs-miRNA-206 (C) and miRNA-210-5p (D) were quantified using RT-qPCR. Comparison of mean miRNA expression in patients with obesity and either low-or high-E2F1 expression was performed using Mann-Whitney non-parametric test. * = Pv < 0.05. (E-G): Inter-correlations of E2F1, miRNA-206, and miRNA-210-5p; miRNA-206 (E) and miRNA-210-5p (F) correlation with adjusted E2F1 expression in human VAT. Intercorrelation between the two miRNAs (G). Shown are data from the two cohorts-cohort 1 participants are depicted in red circles, cohort 2 in black squares. Correlation coefficients (r p ) and statistical significance (Pv) for each cohort separately (Cohort 1, Beer-Sheva, Israel in red; Cohort 2, Leipzig, Germany in black) and for the joint cohort (in bold black) were analyzed using Pearson's correlation test.
## E2f1 regulation of mirna-206 and mirna-210-5p expression
To determine if E2F1 may be a direct transcriptional regulator of the two miRNAs, we first conducted a prediction analysis of putative E2F1 binding sites in the minimal promoter regions (−1000 from the transcription initiation site) of miRNA-206 and 210-5p. This DNA sequence analysis suggested a high probability for four and 10 putative binding sites for E2F1 in minimal promoter regions up-stream of miRNA-206 and 210-5p, respectively (data not shown). To gain experimental support for E2F1 binding to potential regulatory regions of the two miRNAs, we searched for publicly available E2F1 ChIP-seq databases in noncancerous as well as in cancer cells. In all cells tested, the E2F1 ChIP signal was readily noted for the classical E2F1 target gene CDK1 [fig_ref] Figure 5: E2F1 regulates miRNA-206 and miRNA-210 expression [/fig_ref]. For miRNA-210, the non-cancerous mammary gland and mesenchymal stem cells exhibit enrichment of E2F1 in this locus, suggesting that this miRNA may be a direct target of E2F1 [fig_ref] Figure 5: E2F1 regulates miRNA-206 and miRNA-210 expression [/fig_ref] (. Intriguingly, among cancerous cells, the E2F1 ChIP signal was present for some cells (melanoma, myeloma, and MDA-MB-231 breast cancer cells), but not for others (HeLa, Burkitt lymphoma, MCF7 and K562) [fig_ref] Figure 5: E2F1 regulates miRNA-206 and miRNA-210 expression [/fig_ref]. In contrast, the E2F1 ChIP signal was seemingly absent in all cell types for miRNA-206, suggesting that if regulated by E2F1, the mode of regulation is likely indirect [fig_ref] Figure 5: E2F1 regulates miRNA-206 and miRNA-210 expression [/fig_ref]. To further support experimentally the regulation of the miR-206 and 210-5p expression by E2F1, we manipulated E2F1 expression in HEK-293 cells and tested the effect on the expression level of both miRNAs. E2F1 knock-down using siRNA (confirmed using this protocol elsewhere [bib_ref] A TRAIL-TL1A Paracrine Network Involving Adipocytes, Macrophages, and Lymphocytes Induces Adipose Tissue..., Maixner [/bib_ref] resulted in significant downregulation of both miRNA-206 and miRNA-210-5p [fig_ref] Figure 5: E2F1 regulates miRNA-206 and miRNA-210 expression [/fig_ref] , while the over-expression of E2F1 resulted in a trend of up-regulation for both miRNAs [fig_ref] Figure 5: E2F1 regulates miRNA-206 and miRNA-210 expression [/fig_ref]. Jointly, these results reinforce the hypothesis that miR-206 and miR-210-5p are up-regulated down-stream of E2F1 in VAT in human obesity.
tively (data not shown). To gain experimental support for E2F1 binding to potential regulatory regions of the two miRNAs, we searched for publicly available E2F1 ChIP-seq databases in non-cancerous as well as in cancer cells. In all cells tested, the E2F1 ChIP signal was readily noted for the classical E2F1 target gene CDK1 [fig_ref] Figure 5: E2F1 regulates miRNA-206 and miRNA-210 expression [/fig_ref]. For miRNA-210, the non-cancerous mammary gland and mesenchymal stem cells exhibit enrichment of E2F1 in this locus, suggesting that this miRNA may be a direct target of E2F1 [fig_ref] Figure 5: E2F1 regulates miRNA-206 and miRNA-210 expression [/fig_ref]. Intriguingly, among cancerous cells, the E2F1 ChIP signal was present for some cells (melanoma, myeloma, and MDA-MB-231 breast cancer cells), but not for others (HeLa, Burkitt lymphoma, MCF7 and K562) [fig_ref] Figure 5: E2F1 regulates miRNA-206 and miRNA-210 expression [/fig_ref]. In contrast, the E2F1 ChIP signal was seemingly absent in all cell types for miRNA-206, suggesting that if regulated by E2F1, the mode of regulation is likely indirect [fig_ref] Figure 5: E2F1 regulates miRNA-206 and miRNA-210 expression [/fig_ref]. To further support experimentally the regulation of the miR-206 and 210-5p expression by E2F1, we manipulated E2F1 expression in HEK-293 cells and tested the effect on the expression level of both miRNAs. E2F1 knock-down using siRNA (confirmed using this protocol elsewhere [bib_ref] A TRAIL-TL1A Paracrine Network Involving Adipocytes, Macrophages, and Lymphocytes Induces Adipose Tissue..., Maixner [/bib_ref] resulted in significant downregulation of both miRNA-206 and miRNA-210-5p [fig_ref] Figure 5: E2F1 regulates miRNA-206 and miRNA-210 expression [/fig_ref] , while the over-expression of E2F1 resulted in a trend of up-regulation for both miRNAs [fig_ref] Figure 5: E2F1 regulates miRNA-206 and miRNA-210 expression [/fig_ref]. Jointly, these results reinforce the hypothesis that miR-206 and miR-210-5p are up-regulated down-stream of E2F1 in VAT in human obesity. to 80% confluence in 6 well plates and subjected to E2F1 knockdown using either non-coding siRNA (si_NS) or anti-E2F1 siRNA (si_E2F1), incubated for 48h, then snap-frozen and processed for miRNA purification. Expression levels of miRNA-206 and miRNA-210-5p were measured using qRT-PCR. Presented are results of four experiments, each with two biological replicates and two technical replicates. (E). Cells were transfected with plasmids encoding either "empty" pCMV plasmid or a pCMV-hE2F1 plasmid, incubated for 48h, then snap-frozen and processed for miRNA isolation. Expression levels of miRNA-206 and miRNA-210-5p were measured using qRT-PCR. Presented are results of three experiments, each with two biological replicates and two technical replicates. Results are mean ± SEM. ** = Pv ≤ 0.01. Comparison of means was done using Mann-Whitney non-parametric test. Effective E2F1 (D) HEK-293 cells were grown to 80% confluence in 6 well plates and subjected to E2F1 knockdown using either non-coding siRNA (si_NS) or anti-E2F1 siRNA (si_E2F1), incubated for 48h, then snap-frozen and processed for miRNA purification. Expression levels of miRNA-206 and miRNA-210-5p were measured using qRT-PCR. Presented are results of four experiments, each with two biological replicates and two technical replicates. (E). Cells were transfected with plasmids encoding either "empty" pCMV plasmid or a pCMV-hE2F1 plasmid, incubated for 48h, then snap-frozen and processed for miRNA isolation. Expression levels of miRNA-206 and miRNA-210-5p were measured using qRT-PCR. Presented are results of three experiments, each with two biological replicates and two technical replicates. Results are mean ± SEM. ** = Pv ≤ 0.01. Comparison of means was done using Mann-Whitney non-parametric test. Effective E2F1 knock-down/over-expression using the exact siRNA/plasmid transfection protocol was demonstrated elsewhere (1).
## Mirna-206 and mirna-210-5p expression in vat may link high vat-e2f1 t2dm in patients with severe obesity
Next, we sought to assess whether miR-206 and miR-210-5p could function as molecular mediators of E2F1, linking high-VAT expression of E2F1 with a dysmetabolic phenotype of obesity. We first examined whether the combination of miR-206 and 210-5p had any known associations with obesity-related disease. Using the miEAA online tool, we discovered that the miRNA-206/miRNA-210-5p "duo" was over-represented in the scientific literature in relation to a set of diseases associated with obesity, including, among others, lipid disorders, elevated glucose levels and diabetes complications, non-alcoholic fatty liver disease, as well as vascular calcification and atherosclerosis, all evolving from disrupted metabolic function. To gain further insight on possible mechanisms for the involvement of miR-206 and miR-210-5p in metabolic disease, we assessed their association with participants' metabolic markers, specifically circulating lipids and blood glucose levels. To be comparable among the two cohorts, we included only patients with obesity in cohort 1. We did not detect significant associations between the two E2F1-related miRNAs and glycemic or lipid parameters in cohort 1 [fig_ref] Table 3: Correlations between glycemic and circulating lipids parameters and miRNA-206 and miRNA-210-5p in... [/fig_ref]. Yet, in cohort 2, in patients with extreme obesity and in whom high-VAT-E2F1 was associated with T2DM, while lipid profile was not associated with miRNA-206 or miRNA-210-5p expression [fig_ref] Table 4: Correlations between glycemic and circulating lipids parameters and miRNA-206 and miRNA-210-5p in... [/fig_ref] , significant correlations were observed between the miRNAs and glycemic parameters (FPG, FPI): miRNA-206 correlated with FPG levels and miR-210-5p correlated with both FPG and fasting plasma insulin (FPI) levels (Table 4, [fig_ref] Figure 6: Expression of miRNA-206 and miRNA-210-5p in human VAT associates with T2DM in... [/fig_ref] -C, respectively), signifying a systemic glucose-intolerant/insulin-resistant profile. In accordance, by group analysis, those with high-VAT-E2F1 and T2DM exhibited a significantly higher average of miR-210-5p levels (Pv = 0.024), and a nearly significant trend for higher miR-206 levels (Pv = 0.058) [fig_ref] Figure 6: Expression of miRNA-206 and miRNA-210-5p in human VAT associates with T2DM in... [/fig_ref].
# Discussion
E2F1 is a well-studied transcription factor that has been shown to be central in multiple biological networks. E2F1-mediated regulation of gene expression involves several molecular mechanisms, including direct binding to target gene promoters, resulting in transcriptional activation/repression and indirect mechanisms such as via miRNAs. Mostly studied in the context of cell-cycle regulation, a cancer-related E2F1-miRNA network has been documented. In it, E2F1 regulates the transcription of certain miRNAs, which in turn regulates their targets, E2F1 itself being one. Beyond its roles in cell-cycle control, E2F1's involvement in regulating metabolism has been studied in key metabolic organs including the pancreatic beta cells [bib_ref] Impaired pancreatic growth, β cell mass, and β cell function in E2F1..., Fajas [/bib_ref] [bib_ref] The CDK4-pRB-E2F1 pathway controls insulin secretion, Annicotte [/bib_ref] , liver [bib_ref] E2F1 promotes hepatic gluconeogenesis and contributes to hyperglycemia during diabetes, Giralt [/bib_ref] [bib_ref] E2F1 mediates sustained lipogenesis and contributes to hepatic steatosis, Denechaud [/bib_ref] [bib_ref] E2F1 inhibits circulating cholesterol clearance by regulating Pcsk9 expression in the liver, Lai [/bib_ref] , and adipose tissue. In the latter, our previous studies placed E2F1 at a central hub within a molecular network, regulating adipocyte autophagy, MAPK stress-signaling, and gene expression of TNF superfamily members [bib_ref] ASK1 (MAP3K5) is transcriptionally upregulated by E2F1 in adipose tissue in obesity,..., Haim [/bib_ref] [bib_ref] Elevated autophagy gene expression in adipose tissue of obese humans: A potential..., Haim [/bib_ref] [bib_ref] A TRAIL-TL1A Paracrine Network Involving Adipocytes, Macrophages, and Lymphocytes Induces Adipose Tissue..., Maixner [/bib_ref]. Each of these down-stream effectors of E2F1 within adipose tissue only partially mediates E2F1's putative role in linking obesity with a dysmetabolic obesity phenotype. In the present study, we demonstrate, using two independent cohorts, that in adipose tissue in obesity, an E2F1-miRNA network distinct from that shown in cancer may operate. miRNA-206 and miRNA-210-5p were elevated in VAT of patients with obesity in association with E2F1, and were not altered between patients without vs. with obesity but with comparable E2F1 levels. In two separate populations, one of which (cohort 2) included patients with extreme obesity, in which high VAT-E2F1 corresponded
# Discussion
E2F1 is a well-studied transcription factor that has been shown to be central in multiple biological networks. E2F1-mediated regulation of gene expression involves several molecular mechanisms, including direct binding to target gene promoters, resulting in transcriptional activation/repression and indirect mechanisms such as via miRNAs. Mostly studied in the context of cell-cycle regulation, a cancer-related E2F1-miRNA network has been documented. In it, E2F1 regulates the transcription of certain miRNAs, which in turn regulates their targets, E2F1 itself being one. Beyond its roles in cell-cycle control, E2F1's involvement in regulating metabolism has been studied in key metabolic organs including the pancreatic beta cells [bib_ref] Impaired pancreatic growth, β cell mass, and β cell function in E2F1..., Fajas [/bib_ref] [bib_ref] The CDK4-pRB-E2F1 pathway controls insulin secretion, Annicotte [/bib_ref] , liver [bib_ref] E2F1 promotes hepatic gluconeogenesis and contributes to hyperglycemia during diabetes, Giralt [/bib_ref] [bib_ref] E2F1 mediates sustained lipogenesis and contributes to hepatic steatosis, Denechaud [/bib_ref] [bib_ref] E2F1 inhibits circulating cholesterol clearance by regulating Pcsk9 expression in the liver, Lai [/bib_ref] , and adipose tissue. In the latter, our previous studies placed E2F1 at a central hub within a molecular network, regulating adipocyte autophagy, MAPK stress-signaling, and gene expression of TNF superfamily members [bib_ref] ASK1 (MAP3K5) is transcriptionally upregulated by E2F1 in adipose tissue in obesity,..., Haim [/bib_ref] [bib_ref] Elevated autophagy gene expression in adipose tissue of obese humans: A potential..., Haim [/bib_ref] [bib_ref] A TRAIL-TL1A Paracrine Network Involving Adipocytes, Macrophages, and Lymphocytes Induces Adipose Tissue..., Maixner [/bib_ref]. Each of these down-stream effectors of E2F1 within adipose tissue only partially mediates E2F1's putative role in linking obesity with a dysmetabolic obesity phenotype. In the present study, we demonstrate, using two independent cohorts, that in adipose tissue in obesity, an E2F1-miRNA network distinct from that shown in cancer may operate. miRNA-206 and miRNA-210-5p were elevated in VAT of patients with obesity in association with E2F1, and were not altered between patients without vs. with obesity but with comparable E2F1 levels. In two separate populations, one of which (cohort 2) included patients with extreme obesity, in which high VAT-E2F1 corresponded to obesity complicated by T2DM, expression levels of both miRNAs intercorrelated, each also with E2F1 expression itself. Indeed, the possibility of direct (for miRNA-210-5p) or indirect (for miRNA-206) regulation by E2F1 in different cell types was proposed by mining deposited E2F1 ChIP datasets, and by experimentally demonstrating that siRNA-mediated E2F1 knockdown significantly decreased expression, and E2F1 over-expression trended to over-express both miRNA-206 and 210-5p. Finally, consistent with scientific literature linking these miRNAs with a range of metabolic diseases, we observed in patients with extreme obesity, that VAT expression of these miRNAs correlated with T2DM. Overall, our data suggest that E2F1 up-regulates the expression of miRNA-206 and 210-5p in human VAT, situating the E2F1-miRNA-206/210-5p pathway as an addition to an intricate E2F1-regulated network in visceral adipose tissue, which may contribute to an obesity phenotype complicated by T2DM.
Although, to the best of our knowledge, the E2F1-mediated regulation of miRNA-206 and miRNA-210-5p is a novel finding, our results are supported by substantial literature on the physiological functions of both miRNAs, which seem to substantially overlap with those of E2F1. In particular, miRNA-210 has been reported to function as a major "hypoxi-amiR", hypoxia-inducible miRNA in a wide range of cells, serving to adjust the cells to hypoxic conditions. In this context, miRNA-210 was shown to alter cellular metabolism by compromising mitochondrial function and limiting glycolytic-to-mitochondrial substrate transition via targeting components of the Krebs cycle and electron transport chain [bib_ref] Hypoxia-regulated microRNA-210 modulates mitochondrial function and decreases ISCU and COX10 expression, Chen [/bib_ref] [bib_ref] Distinct Effects of miR-210 Reduction on Neurogenesis: Increased Neuronal Survival of Inflammation..., Voloboueva [/bib_ref] [bib_ref] MIR-210 modulates mitochondrial respiration in placenta with preeclampsia, Muralimanoharan [/bib_ref]. Concordantly, cells transfected with miRNA-210 demonstrated decreased oxygen consumption and a greater dependence on glycolysis for energy metabolism [bib_ref] MIR-210 modulates mitochondrial respiration in placenta with preeclampsia, Muralimanoharan [/bib_ref]. Indeed, recent studies indicate that exosomal miRNA-210 may disrupt adipocyte glucose mitochondrial oxidation and inhibit the browning of naïve adipocytes [bib_ref] MiR-210 in Exosomes Derived from Macrophages under High Glucose Promotes Mouse Diabetic..., Tian [/bib_ref] [bib_ref] Adipose-derived exosomal miR-210/92a cluster inhibits adipose browning via the FGFR-1 signaling pathway..., Zhang [/bib_ref]. Remarkably, E2F1 was repeatedly demonstrated to inhibit oxidative metabolism via inhibition of metabolic flux to the mitochondria (regulating pyruvate dehydrogenase complex), mitochondrial biogenesis and enzymatic activity [bib_ref] Acute loss of transcription factor E2F1 induces mitochondrial biogenesis in Hela cells, Goto [/bib_ref] [bib_ref] E2F transcription factor-1 regulates oxidative metabolism, Blanchet [/bib_ref] [bib_ref] Regulation of the PDK4 Isozyme by the Rb-E2F1 Complex, Hsieh [/bib_ref] , and was suitably demonstrated to inhibit adipocyte beiging/browning (i.e., drive adipocyte "whitening" [bib_ref] Transcription Factor E2F1 Knockout Promotes Mice White Adipose Tissue Browning Through Autophagy..., Xiong [/bib_ref]. Thus, it is plausible that miR-210 mediates E2F1-dependent adipose tissue whitening in obesity.
A similar E2F1-mediatory role for miRNA-206 can be envisioned, as miRNA-206 was shown to interfere with intracellular glucose utilization and increased E2F1 expression in human VAT associated with decreased systemic glucose infusion rate during hyperinsulinemic-euglycemic clamp studies [bib_ref] Elevated autophagy gene expression in adipose tissue of obese humans: A potential..., Haim [/bib_ref]. In pancreatic beta-cells, miRNA-206 directly inhibits glucokinase(GK) activity via a conserved binding site at the 3 -UTR of this gene [bib_ref] MiR-206 is expressed in pancreatic islets and regulates glucokinase activity, Vinod [/bib_ref] [bib_ref] Expression of miR-206 in human islets and its role in glucokinase regulation, Joglekar [/bib_ref] , thereby interfering with glucose-stimulated insulin secretion. Interestingly, miRNA-206 expression in beta-cells was shown to increase upon high-fat feeding [bib_ref] MiR-206 is expressed in pancreatic islets and regulates glucokinase activity, Vinod [/bib_ref]. Beyond its effect on GK, which is a beta-cell/hepatocyte-specific enzyme, miRNA-206 similarly regulates hexokinase(HK) in malignant cells [bib_ref] miR-206 regulates non-small-cell lung cancer cell aerobic glycolysis by targeting hexokinase, Jia [/bib_ref]. miRNA-206-driven inhibition was also noted on glucose-6-phosphate dehydrogenase(G6PD) [bib_ref] MicroRNA-206 regulates cell proliferation by targeting G6PD in skeletal muscle, Jiang [/bib_ref] and on the end-glycolytic enzyme pyruvate kinase(PK) [bib_ref] A novel miR-206/hnRNPA1/PKM2 axis reshapes the Warburg effect to suppress colon cancer..., Fu [/bib_ref] , decelerating hexose monophosphate shunt and glycolytic flux, and thereby, glucose handling. Indeed, several separate reports demonstrated that overexpression of miRNA-206 results in decreased glucose uptake into a wide variety of cells, and vice-versa, its elimination resulted in improved glucose handling [bib_ref] MiR-206 is expressed in pancreatic islets and regulates glucokinase activity, Vinod [/bib_ref] [bib_ref] miR-206 regulates non-small-cell lung cancer cell aerobic glycolysis by targeting hexokinase, Jia [/bib_ref] [bib_ref] A novel miR-206/hnRNPA1/PKM2 axis reshapes the Warburg effect to suppress colon cancer..., Fu [/bib_ref]. Albeit not directly studied so far in adipose tissue, our results propose that miR-206 could similarly operate in adipocytes down-stream of E2F1, acting as a mediator of E2F1-associated dysglycemia.
As mentioned above, a growing body of literature implicates E2F1 in the regulation of metabolism in key metabolic organs: by up-regulating PDH kinase 4(PDK4), which phosphorylates and inhibits the PDH complex, E2F1 supports glycolysis over glucose oxidation in myocytes [bib_ref] Regulation of the PDK4 Isozyme by the Rb-E2F1 Complex, Hsieh [/bib_ref]. In hepatocytes, up-regulated E2F1 drives lipid accumulation and gluconeogenesis, thus, promoting hepatic steatosis and systemic hyperglycemia [bib_ref] E2F1 promotes hepatic gluconeogenesis and contributes to hyperglycemia during diabetes, Giralt [/bib_ref] [bib_ref] E2F1 mediates sustained lipogenesis and contributes to hepatic steatosis, Denechaud [/bib_ref] [bib_ref] E2F1 inhibits circulating cholesterol clearance by regulating Pcsk9 expression in the liver, Lai [/bib_ref]. In pancreatic β-cells, it regulates the expression of Kir6.2, driving glucose-stimulated insulin secretion and supporting the hyper-insulinemic response [bib_ref] Impaired pancreatic growth, β cell mass, and β cell function in E2F1..., Fajas [/bib_ref] [bib_ref] The CDK4-pRB-E2F1 pathway controls insulin secretion, Annicotte [/bib_ref]. In the adipocyte, as mentioned, it promotes "whitening" (=inhibits beiging) [bib_ref] Transcription Factor E2F1 Knockout Promotes Mice White Adipose Tissue Browning Through Autophagy..., Xiong [/bib_ref]. In accordance, in E2F1-KO mice, despite the decreased pancreatic size (attributed to impaired postnatal growth), mice were not diabetic due to concurrent increased adipose browning and insulin sensitivity [bib_ref] Impaired pancreatic growth, β cell mass, and β cell function in E2F1..., Fajas [/bib_ref]. The latter phenotype is consistent with human data implicating E2F1's role in the generation of systemic insulin resistance when up-regulated in obesity. E2F1 is also mechanistically related to inflammation: both acute inflammatory stimuli (TPA, arsenate) [bib_ref] Selective activation of NF-κB and E2F by low concentration of arsenite in..., Yamamoto [/bib_ref] [bib_ref] Identification of the Rage-dependent gene regulatory network in a mouse model of..., Riehl [/bib_ref] and chronic inflammation [bib_ref] Chronic Inflammation Promotes Retinoblastoma Protein Hyperphosphorylation and E2F1 Activation, Ying [/bib_ref] induce E2F1 protein and mRNA expression and activate the retinoblastoma-E2F1 axis, respectively. We have recently demonstrated that in adipose tissue, lymphocytes-derived TL1A(TNFSF15) can directly induce the adipose tissue E2F1 expression, which in turn stimulates the expression of adipose tissue TRAIL(TNFSF10) that further induces TL1A, generating a feed-forward, E2F1-inflammatory cytokine feedback loop [bib_ref] A TRAIL-TL1A Paracrine Network Involving Adipocytes, Macrophages, and Lymphocytes Induces Adipose Tissue..., Maixner [/bib_ref]. In all of these sites and mechanisms, the respective roles of E2F1-induced miRNA-206 and 210-5p remain to be discovered, as is the potential beneficial effects of antagonizing these E2F1-effector miRNAs. Our results suggest that this approach may prove beneficial, particularly in dysglycemic persons with obesity and high-VAT-E2F1 expression.
There are a few strengths, but also limitations to our study, which warrant consideration. Paralleling the "general" reproducibility crisis in bio-medical research, studies on miRNAs have particularly seen low reproducibility among cohorts. Hence, our study is strengthened by insisting on results' validation using two independent, carefully constructed cohorts, from two centers coordinated by the pre-analytic procedures (human tissue sampling and handling). Cohort 2 represents a more extreme obesity phenotype, with older patients in whom high VAT-E2F1 is uniformly associated with obesity being complicated by T2DM. Moreover, to ensure that results do not merely depend on specific methodological approaches, VAT-E2F1 expression was assessed at the protein level in cohort 1, and at the mRNA level in cohort 2. Similarly, the determination of miRNAs was performed using Nanostring© or rtPCR in cohorts 1 and 2, respectively. The statistical approach undertaken also allows us to carefully predict the chance that our results are random by considering each cohort separately, as well as when joined into a fused cohort. An additional strength of our study is the experimental confirmation of bioinformatic predictions, particularly the regulation of the two miRNAs by manipulating E2F1 levels. Yet, the experimental model we used for miRNA screening consisted of whole adipose tissue, while the origin of VAT-E2F1 is likely the adipocytes [bib_ref] Elevated autophagy gene expression in adipose tissue of obese humans: A potential..., Haim [/bib_ref]. We therefore cannot rule out that our approach "dilutes" or masks adipocyte-specific processes. Thus, we may have missed adipocyte-only E2F1-related miRNAs consistent with the cancer-associated E2F1-miRNA network, which we initially anticipated. It is also worth noting that the mechanistic confirmation provided here for E2F1-regulated expression of miRNA-206 and miRNA-210-5p is demonstrated in HEK293 cells rather than adipocytes. This is due to the well-known difficulty of genetically manipulating mature adipocytes [bib_ref] High Efficiency Lipid-Based siRNA Transfection of Adipocytes in Suspension, Kilroy [/bib_ref] , with human mature adipocytes being an even greater challenge. Yet, HEK-293 cells are of human origin, gain-and loss-of-function approaches yielded mirroring results, and were combined with cross-sectional analyses of human adipose tissue, supporting our findings.
In conclusion, this study expands our understanding of the E2F1 molecular network that operates in human visceral adipose tissue in obesity, and how it associates with extreme dysmetabolic obesity complicated by T2DM (Scheme 1). Differing from our hypothesis we found no evidence for a functional E2F1-miRNA network reminiscent of that which was described in cancer (the E2F1-miRNA cancer-progression network). Yet, in human visceral adipose tissue, we proposed that in addition to E2F1-mediated regulation of ASK1 (MAP3K5) MAP kinase signaling pathway, autophagy, and TNF superfamily members, E2F1 regulates miRNA 206 and 210-5p. In light of this study's objectives, we demonstrate that in extreme obesity setting these possibly link high-VAT-E2F1 with T2DM and systemic insulin resistance. Thus, the importance of the study is: i. expanding basic pathophysiological understanding of the molecular mediators that link high E2F1 in VAT to a metabolically complicated extreme obesity; ii. given that miRNA-based molecules are potential molecules for drug development, our findings propose miRNA-206 and 210-5p as putative new targets for alleviating obesity-associated dysglycemia. Data Availability Statement: All data will be made available by request to the corresponding authors.
## Conflicts of interest: no potential conflict of interest is reported by the authors.
## Abbreviations
[fig] Figure 1: Figure 1. Two cohorts of matched patients with high-vs. low-E2F1 levels in VAT. Cohort 1: Human adipose tissue E2F1 protein expression range was determined on a pre-screened cohort of n = 67 persons, and high and low expression were those in the upper and lower two quintiles (40%), respectively. Based on this definition, cohort 1 (Beer-Sheva, Israel), consisted of age, sex, and (when relevant) BMI-matched triplets, each consisting of patients "without obesity and with low-E2F1 expression", "with obesity and low-E2F1 expression" and "with obesity and high-E2F1 expression". The matching of eight triplets of patients (i.e., n = 8 × 3 = 24) for E2F1 expression (A), BMI (B), and age (C) is shown. Cohort 2 (Leipzig, Germany) consisted of 10 similarly matched patients, with the following differences: E2F1 expression definition and matching; (D) was based on mRNA expression, and the cohort included pairs only of patients with extreme obesity (BMI ≥ 45 kg/m 2 ) also matched for BMI (E) and age (F). Different colors represent each matched couple/triplet. Dots represent individual patients. *** = Pv < 0.001, **** = Pv < 0.0001, ns = non-significant. [/fig]
[fig] Figure 3: Members of the cancer-associated miRNA-E2F1 co-regulatory network are not altered in association with E2F1 in human VAT: (A) Illustration of the cancer-related miRNA-E2F1 co-regulation network. Expression level of miRNA106a-17-5p (B) and miRNA-106b (C) derived from nanos-tring© results of cohort 1, comparing patients with obesity and low-vs. high-E2F1 expression. (D-F) Quantitative rt-PCR results of VAT expression of miRNA-17-5p, miRNA-106a, and miRNA-106b, respectively, comparing patients with obesity and low-vs. high-E2F1 expression from cohort 2. Mean expressions were compared using Mann-Whitney non-parametric test. Circles and squares represent individual values, horizontal lines represent mean. All comparisons were statistically non-significant (Pv > 0.05). [/fig]
[fig] Figure 4: miRNA-206 and miRNA-210-5p intercorrelate and are candidate E2F1 targets. (A-D): [/fig]
[fig] Figure 5: E2F1 regulates miRNA-206 and miRNA-210 expression. (A-C). E2F1 ChIP-seq results in multiple non-cancerous and cancer cells mined from publicly available databases (see Methods for detail). Depicted are genomic regions in the vicinity of CDK1-a classical E2F1 target gene (A), miRNA-210 (B), and miRNA-206 (C). (D) HEK-293 cells were grown [/fig]
[fig] Figure 6: Expression of miRNA-206 and miRNA-210-5p in human VAT associates with T2DM in extreme obesity. (A-C). Correlation between expression levels of miRNA-206 and miRNA-210-5p (by rt-PCR, cohort 2) with fasting plasma glucose (FPG) or fasting plasma insulin (FPI). Correlations were performed using Pearson's test. (D-E). Group comparison of Cohort 2: participants were divided by diabetes status (w/o T2DM = low VAT-E2F1; with T2DM = high VAT-E2F1), and mean expression levels of miRNA-206 and 210-5p were compared between the two groups using Mann-Whitney non-parametric test. [/fig]
[table] Table 1: Clinical characteristics of participants in cohort 1. [/table]
[table] Table 2: Clinical characteristics of participants in cohort 2. [/table]
[table] Table 3: Correlations between glycemic and circulating lipids parameters and miRNA-206 and miRNA-210-5p in cohort 1. [/table]
[table] Table 4: Correlations between glycemic and circulating lipids parameters and miRNA-206 and miRNA-210-5p in cohort 2. [/table]
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Applications of Ultrasound-Mediated Gene Delivery in Regenerative Medicine
# Introduction
The field of regenerative medicine was established with the goal of regrowing human tissues to restore endogenous function following traumatic injury or disease [bib_ref] The Progression of Regenerative Medicine and its Impact on Therapy Translation, Jacques [/bib_ref]. The global popularity of this interdisciplinary field has resulted in a paradigm shift from palliation to an emphasis on restorative therapies [bib_ref] Transforming healthcare through regenerative medicine, Jessop [/bib_ref]. Gene delivery, the administration of genetic material to modify gene expression, presents a promising approach to accomplish this goal across various disease states. However, identifying safe delivery vectors capable of producing a sustained biological effect, crucial for clinical implementation and success, remains a major challenge to this day [bib_ref] Gene delivery in tissue engineering and regenerative medicine, Fang [/bib_ref] [bib_ref] New Tools in Regenerative Medicine: Gene Therapy, Ruiz [/bib_ref].
There are two fundamental gene delivery systems: viral and non-viral [bib_ref] Gene transfer strategies in tissue engineering, Bleiziffer [/bib_ref]. Viral vectors utilize the ability of viruses to introduce their DNA into host cells, a process called transduction. While capable of successfully yielding gene expression due to the ability of the viral structure to prevent degradation, several studies have demonstrated that the use of these carriers presents several limitations, including immunogenicity [bib_ref] Recent advances in the development of gene delivery systems, Sung [/bib_ref] , off-target delivery [bib_ref] Engineering targeted viral vectors for gene therapy, Waehler [/bib_ref] , and difficult vector production [bib_ref] Viral vectors: From virology to transgene expression, Bouard [/bib_ref]. Despite advances to improve the safety of viral vectors [bib_ref] Viral vector-mediated transgenic cell therapy in regenerative medicine: Safety of the process, Liu [/bib_ref] [bib_ref] Current Trends in Viral Gene Therapy for Human Orthopaedic Regenerative Medicine, Venkatesan [/bib_ref] [bib_ref] Suicide gene approach using a dualexpression lentiviral vector to enhance the safety..., Alaee [/bib_ref] , the development of effective non-viral delivery systems is still needed [bib_ref] Viral and nonviral delivery systems for gene delivery, Nayerossadat [/bib_ref]. [bib_ref] Bone Regeneration Using Gene-Activated Matrices, D'mello [/bib_ref] Magnetofection Magnetic particles complexed with DNA and an external magnetic field
Fast delivery of nucleic acids, high transduction efficiency, low-dose requirements Localization can be difficult in vivo, particle size impacts cell entry, cytotoxicity [bib_ref] Recent advances in the development of gene delivery systems, Sung [/bib_ref] Electroporation High voltage electric pulses to increase membrane permeability High throughput, low cost, more efficient than naked DNA injection or sonoporation Variable transfection efficiency, limited cell viability, non-homogenous tissue regeneration, potential tissue damage [bib_ref] Non-viral Gene Delivery Methods for, Gantenbein [/bib_ref] [bib_ref] Ultrasound-based nonviral gene delivery induces bone formation in vivo, Sheyn [/bib_ref] Sonoporation Ultrasound waves create pores in cell membrane due to cavitation Noninvasive, less tissue damage compared to electroporation, ultrasound is highly accepted in the clinical setting, more efficient than naked DNA injection, systemic injection is possible
Low transfection efficiency, cell membrane damage is possible, low reproducibility [bib_ref] Sonoporation: Gene transfer using ultrasound, Tomizawa [/bib_ref] Within the last two decades, the use of sonoporation-based gene delivery for tissue regeneration has gained traction, and while promising results have been previously demonstrated, further improvements are still being made. In this review, we will focus on the use of sonoporation for tissue regeneration and outline the in vivo studies conducted thus far.
## Sonoporation-based gene delivery
Sonoporation has been shown to be an effective non-viral gene delivery system in both in vitro and in vivo studies across many organ systems [bib_ref] Sonoporation: Gene transfer using ultrasound, Tomizawa [/bib_ref] [bib_ref] Acoustofluidic sonoporation for gene delivery to human hematopoietic stem and progenitor cells, Belling [/bib_ref] [bib_ref] Nucleic acid delivery with microbubbles and ultrasound, Rychak [/bib_ref]. In comparison to some nonviral methods, however, sonoporation is noted as having lower gene transfer efficiency and may also cause cells to undergo apoptosis due to damage to the cell membrane [bib_ref] Sonoporation: Gene transfer using ultrasound, Tomizawa [/bib_ref]. The use of ultrasound energy to enhance gene delivery into targeted cells was first evaluated in the 1980s [bib_ref] Transfection of mammalian cells with plasmid DNA by scrape loading and sonication..., Fechheimer [/bib_ref]. Ultrasound waves can modify the permeability of the cell plasma membrane, and this characteristic can be applied to gene delivery. In order to enhance the efficiency of sonoporation-based gene delivery, the use of ultrasound contrast agents, microbubbles, was explored and has since become quite commonly used. The ultrasonic waves induce cavitation, the growth, oscillation, and collapse of small gas bubbles in a fluid, which was Bioengineering 2022, [bib_ref] Viral vector-mediated transgenic cell therapy in regenerative medicine: Safety of the process, Liu [/bib_ref] 3 of 17 first visualized in 1999 using scanning electron microscopy [bib_ref] Induction of cell-membrane porosity by ultrasound, Tachibana [/bib_ref]. Cavitation results in greater permeabilization of cell membranes, allowing for nucleic acids to passively diffuse into the cytoplasm through cavitation-induced pores [fig_ref] Figure 1: Image depicting the induction of a pore in the cell membrane by... [/fig_ref]. Moreover, the use of microbubbles enables real-time monitoring of the sonoporation process on the ultrasound screen.
first evaluated in the 1980s [bib_ref] Transfection of mammalian cells with plasmid DNA by scrape loading and sonication..., Fechheimer [/bib_ref]. Ultrasound waves can modify the permeability of the cell plasma membrane, and this characteristic can be applied to gene delivery. In order to enhance the efficiency of sonoporation-based gene delivery, the use of ultrasound contrast agents, microbubbles, was explored and has since become quite commonly used. The ultrasonic waves induce cavitation, the growth, oscillation, and collapse of small gas bubbles in a fluid, which was first visualized in 1999 using scanning electron microscopy [bib_ref] Induction of cell-membrane porosity by ultrasound, Tachibana [/bib_ref]. Cavitation results in greater permeabilization of cell membranes, allowing for nucleic acids to passively diffuse into the cytoplasm through cavitation-induced pores [fig_ref] Figure 1: Image depicting the induction of a pore in the cell membrane by... [/fig_ref]. Moreover, the use of microbubbles enables real-time monitoring of the sonoporation process on the ultrasound screen. In addition to serving as a contrast agent in medical imaging [bib_ref] Microbubble contrast agents: Targeted ultrasound imaging and ultrasound-assisted drug-delivery applications, Klibanov [/bib_ref] , first approved by the United States Food and Drug Administration (FDA) in 1994 [bib_ref] Safety with Echocardiographic Contrast Agents, Muskula [/bib_ref] , microbubbles have been shown to enhance sonoporation. When used in conjunction with microbubbles, ultrasound-induced cavitation effects increase the efficiency of DNA uptake through cavitation-induced pores. Studies have been conducted in which microbubbles have been excluded, and plasmid DNA was injected with low-intensity pulsed ultrasound (LIPUS) alone [bib_ref] Effect of Nonviral Plasmid Delivered Basic Fibroblast Growth Factor and Low Intensity..., Kaur [/bib_ref] , but transfection was far superior when microbubbles were incorporated [bib_ref] Ultrasound-responsive gene-activated matrices for osteogenic gene therapy using matrix-assisted sonoporation, Nomikou [/bib_ref]. In certain models, without the synergistic effect between microbubbles and ultrasound, effective gene transfer and expression did not occur at all [bib_ref] Ultrasound-mediated gene transfer (sonoporation) in fibrin-based matrices: Potential for use in tissue..., Nomikou [/bib_ref].
Both in vitro and in vivo studies have shown that sonoporation outcomes are significantly impacted by the power settings on the ultrasound, the duration of ultrasound exposure, and the plasmid and microbubbles being injected [bib_ref] Induction of cell-membrane porosity by ultrasound, Tachibana [/bib_ref] [bib_ref] Understanding ultrasound induced sonoporation: Definitions and underlying mechanisms, Lentacker [/bib_ref] [bib_ref] Effect of non-acoustic parameters on heterogeneous sonoporation mediated by single-pulse ultrasound and..., Qin [/bib_ref] [bib_ref] Sonoporation Increases Therapeutic Efficacy of Inducible and Constitutive BMP2/7 In Vivo Gene..., Feichtinger [/bib_ref]. One study found that there was an inverse relationship between microbubble concentration and cell viability [bib_ref] Studies on neutral, cationic and biotinylated cationic microbubbles in enhancing ultrasound-mediated gene..., Nomikou [/bib_ref] , while another suggested that the use of microbubbles during sonoporation was capable of reducing the skeletal muscle damage observed when naked DNA is injected directly into the tissue [bib_ref] Microbubble ultrasound improves the efficiency of gene transduction in skeletal muscle in..., Lu [/bib_ref]. In addition, the type of microbubbles used may impact the In addition to serving as a contrast agent in medical imaging [bib_ref] Microbubble contrast agents: Targeted ultrasound imaging and ultrasound-assisted drug-delivery applications, Klibanov [/bib_ref] , first approved by the United States Food and Drug Administration (FDA) in 1994 [bib_ref] Safety with Echocardiographic Contrast Agents, Muskula [/bib_ref] , microbubbles have been shown to enhance sonoporation. When used in conjunction with microbubbles, ultrasoundinduced cavitation effects increase the efficiency of DNA uptake through cavitation-induced pores. Studies have been conducted in which microbubbles have been excluded, and plasmid DNA was injected with low-intensity pulsed ultrasound (LIPUS) alone [bib_ref] Effect of Nonviral Plasmid Delivered Basic Fibroblast Growth Factor and Low Intensity..., Kaur [/bib_ref] , but transfection was far superior when microbubbles were incorporated [bib_ref] Ultrasound-responsive gene-activated matrices for osteogenic gene therapy using matrix-assisted sonoporation, Nomikou [/bib_ref]. In certain models, without the synergistic effect between microbubbles and ultrasound, effective gene transfer and expression did not occur at all [bib_ref] Ultrasound-mediated gene transfer (sonoporation) in fibrin-based matrices: Potential for use in tissue..., Nomikou [/bib_ref].
Both in vitro and in vivo studies have shown that sonoporation outcomes are significantly impacted by the power settings on the ultrasound, the duration of ultrasound exposure, and the plasmid and microbubbles being injected [bib_ref] Induction of cell-membrane porosity by ultrasound, Tachibana [/bib_ref] [bib_ref] Understanding ultrasound induced sonoporation: Definitions and underlying mechanisms, Lentacker [/bib_ref] [bib_ref] Effect of non-acoustic parameters on heterogeneous sonoporation mediated by single-pulse ultrasound and..., Qin [/bib_ref] [bib_ref] Sonoporation Increases Therapeutic Efficacy of Inducible and Constitutive BMP2/7 In Vivo Gene..., Feichtinger [/bib_ref]. One study found that there was an inverse relationship between microbubble concentration and cell viability [bib_ref] Studies on neutral, cationic and biotinylated cationic microbubbles in enhancing ultrasound-mediated gene..., Nomikou [/bib_ref] , while another suggested that the use of microbubbles during sonoporation was capable of reducing the skeletal muscle damage observed when naked DNA is injected directly into the tissue [bib_ref] Microbubble ultrasound improves the efficiency of gene transduction in skeletal muscle in..., Lu [/bib_ref]. In addition, the type of microbubbles used may impact the resulting gene expression and cell viability. Biotinylated cationic microbubbles appear to be preferable to neutral forms due to their more efficient binding of both cells and nucleic acids, and they also seem to provide protection when the ultrasound power density is increased, although only a few studies have conducted a direct comparison [bib_ref] Ultrasound-mediated gene transfer (sonoporation) in fibrin-based matrices: Potential for use in tissue..., Nomikou [/bib_ref] [bib_ref] Studies on neutral, cationic and biotinylated cationic microbubbles in enhancing ultrasound-mediated gene..., Nomikou [/bib_ref].
Current studies are trying to find a balance between cell viability and gene expression. In attempts to prolong expression in vivo, up to 85 days, it appears that the plasmid being used has a role to play in the duration of gene expression [bib_ref] Optimising ultrasound-mediated gene transfer (sonoporation) in vitro and prolonged expression of a..., Li [/bib_ref] [bib_ref] Sustained Transgene Expression in Intervertebral Disc Cells In Vivo Mediated by Microbubble-Enhanced..., Nishida [/bib_ref]. Studies have found that the delivery of the gene through an implanted scaffold or matrix may enhance sonoporation-induced gene transfection at the target site up to 25-fold [bib_ref] Ultrasound-mediated gene transfer (sonoporation) in fibrin-based matrices: Potential for use in tissue..., Nomikou [/bib_ref]. A scaffold may also attract endogenous progenitor/stem cells to the injury site [bib_ref] In situ bone tissue engineering via ultrasound-mediated gene delivery to endogenous progenitor..., Bez [/bib_ref] [bib_ref] Ultrasound-Mediated Gene Delivery Enhances Tendon Allograft Integration in Mini-Pig Ligament Reconstruction, Bez [/bib_ref] or provide a protective factor for implanted stem cells, the targets of transfection [bib_ref] Ultrasound-mediated gene transfer (sonoporation) in fibrin-based matrices: Potential for use in tissue..., Nomikou [/bib_ref] [bib_ref] Enhancing ultrasound-mediated cell membrane permeabilisation (sonoporation) using a high frequency pulse regime..., Li [/bib_ref]. However, further studies are warranted to determine how to overcome the possible ultrasound wave attenuation caused by the implanted materials [bib_ref] Ultrasound-mediated gene transfer (sonoporation) in fibrin-based matrices: Potential for use in tissue..., Nomikou [/bib_ref] [bib_ref] Naturally and synthetic smart composite biomaterials for tissue regeneration, Perez [/bib_ref].
## Applications of sonoporation for tissue regeneration
The use of sonoporation for tissue regeneration has been characterized in various animal models . . The use of sonoporation for tissue regeneration.
## Regeneration model animal model references
Bone Regeneration Mouse [bib_ref] Ultrasound-based nonviral gene delivery induces bone formation in vivo, Sheyn [/bib_ref] [bib_ref] Ultrasound-responsive gene-activated matrices for osteogenic gene therapy using matrix-assisted sonoporation, Nomikou [/bib_ref] [bib_ref] Sonoporation Increases Therapeutic Efficacy of Inducible and Constitutive BMP2/7 In Vivo Gene..., Feichtinger [/bib_ref] [bib_ref] Osteoinduction by microbubble-enhanced transcutaneous sonoporation of human bone morphogenetic protein-2, Osawa [/bib_ref] Pig [bib_ref] In situ bone tissue engineering via ultrasound-mediated gene delivery to endogenous progenitor..., Bez [/bib_ref] Soft Tissue-Bone Integration Pig [bib_ref] Ultrasound-Mediated Gene Delivery Enhances Tendon Allograft Integration in Mini-Pig Ligament Reconstruction, Bez [/bib_ref] Myocardial Angiogenesis Mouse [bib_ref] Ultrasound-Targeted Gene Delivery Induces Angiogenesis After a Myocardial Infarction in Mice, Fujii [/bib_ref] [bib_ref] Increased local delivery of antagomir therapeutics to the rodent myocardium using ultrasound..., Kwekkeboom [/bib_ref] Rat [bib_ref] Repeated and targeted transfer of angiogenic plasmids into the infarcted rat heart..., Fujii [/bib_ref] [bib_ref] The use of cationic microbubbles to improve ultrasound-targeted gene delivery to the..., Sun [/bib_ref] Peripheral Angiogenesis Rabbit [bib_ref] Development of safe and efficient novel nonviral gene transfer using ultrasound: Enhancement..., Taniyama [/bib_ref] Rat [bib_ref] Therapeutic Arteriogenesis by Ultrasound-Mediated VEGF 165 Plasmid Gene Delivery to Chronically Ischemic..., Leong-Poi [/bib_ref] [bib_ref] Comparison of Gene Delivery Techniques for Therapeutic Angiogenesis: Ultrasound-Mediated Destruction of Carrier..., Kobulnik [/bib_ref] [bib_ref] Sustained Improvement in Perfusion and Flow Reserve After Temporally Separated Delivery of..., Smith [/bib_ref] [bib_ref] Therapeutic Angiogenesis by Ultrasound-Mediated MicroRNA-126-3p Delivery, Cao [/bib_ref] Pancreatic Islet Regeneration Rat [bib_ref] Regeneration of pancreatic islets in vivo by ultrasound-targeted gene therapy, Chen [/bib_ref] [bib_ref] Ectopic transgenic expression of NKX2.2 induces differentiation of adult pancreatic progenitors and..., Chen [/bib_ref] [bib_ref] Transient overexpression of cyclin D2/CDK4/GLP1 genes induces proliferation and differentiation of adult..., Chen [/bib_ref] [bib_ref] In vivo targeted delivery of ANGPTL8 gene for beta cell regeneration in..., Chen [/bib_ref]
## Sonoporation for skeletal tissue regeneration
Despite the remarkable regenerative capacity of bone tissue, there are several instances where biological processes are unable to fully recuperate and repair bone loss, leading to conditions such as nonunion fractures [bib_ref] Bone Regeneration Based on Tissue Engineering Conceptions-A 21st Century Perspective, Henkel [/bib_ref]. Preliminary studies on sonoporation focused on the ultrasound parameters necessary to achieve efficient gene transfection when injecting naked plasmid DNA into skeletal muscle [bib_ref] Development of safe and efficient novel nonviral gene transfer using ultrasound: Enhancement..., Taniyama [/bib_ref]. These parameters were adopted for use in a 2008 study by Sheyn et al., the first in vivo study on the use of sonoporation for skeletal tissue formation [bib_ref] Ultrasound-based nonviral gene delivery induces bone formation in vivo, Sheyn [/bib_ref]. Published less than 10 years after it was demonstrated that sonoporation induces membrane permeabilization, this study found that the intramuscular injection of a plasmid encoding recombinant human bone morphogenic protein-9 (rhBMP-9), mixed with lipid-stabilized microbubbles, resulted in ectopic bone formation in 6 out of 8 mice treated with sonoporation. However, this study compared these results to electroporation and found that all mice that received electroporation experienced ectopic bone formation. When comparing the ectopic bone formed in each treatment group, the bone volume in electroporation-treated mice was significantly larger than the bone generated by sonoporation. Conversely, the bone volume density of the newly formed tissue generated by sonoporation was found to be significantly larger than that formed by electroporation. Even though it was found to be less efficient than electroporation, this study demonstrated that sonoporation was capable of inducing bone regeneration. The authors also indicated that sonoporation did not exhibit the same adverse effects seen with electroporation, namely muscle tissue damage. Other studies have found that while electroporation increased transfection and induced bone regeneration [bib_ref] Targeted Gene-and-host Progenitor Cell Therapy for Nonunion Bone Fracture Repair, Kimelman-Bleich [/bib_ref] , it can be accompanied by other undesirable effects, such as passive involuntary muscle contractions, tissue damage associated with thermal changes at the site of electric pulses, and nonhomogenous tissue regeneration [bib_ref] Gene transfer into muscle by electroporation in vivo, Aihara [/bib_ref] [bib_ref] Induction of dental pulp stem cell differentiation into odontoblasts by electroporation-mediated gene..., Nakashima [/bib_ref].
A year after the initial Sheyn et al. study was published, researchers in Japan utilized a plasmid-based human BMP-2 construct and transcutaneous sonoporation in male mice [bib_ref] Osteoinduction by microbubble-enhanced transcutaneous sonoporation of human bone morphogenetic protein-2, Osawa [/bib_ref]. Sonoporation with lipid microbubbles was repeated at 24-h intervals for up to 7 days. By 21 days after the final ultrasound treatment, cartilage and immature bone were observed in the gastrocnemius muscle, and evaluation of muscle fibers revealed bone matrix with bone marrow that included blood cells and adipocytes. In addition, alkaline phosphatase (ALP) activity increased significantly and the Ca 2+ concentration was higher, indicating that sonoporation had indeed caused osteoinduction. This study demonstrated that repetition of this procedure significantly increased osteoinduction in the target muscle compared to only one session of sonoporation. This is consistent with other studies that have used sonoporation for gene delivery for other disease models, including cancer [bib_ref] Sonoporation-mediated anti-angiogenic gene transfer into muscle effectively regresses distant orthotopic tumors, Liao [/bib_ref]. It is important to note, however, that repeated intramuscular injection of naked plasmid DNA can cause various degrees of muscle damage and inflammatory responses, even when not applying ultrasound, and thus, repeated injections may introduce additional risks [bib_ref] Inflammatory responses following direct injection of plasmid DNA into skeletal muscle, Mcmahon [/bib_ref].
In an attempt to increase the effectiveness of sonoporation, the use of a matrix, referred to as matrix-assisted sonoporation (MAS), was evaluated by Nomikou et al. [bib_ref] Ultrasound-responsive gene-activated matrices for osteogenic gene therapy using matrix-assisted sonoporation, Nomikou [/bib_ref]. Published in 2018, the researchers utilized an advanced ultrasound-responsive gene-activated matrix (GAM). This matrix, injected into the hind leg of mice, was composed of fibrin and collagen and contained polymeric microbubbles, pVAX-BMP2/7 co-expression plasmids, and C2C12 mouse myoblast precursor cells. Following external application of the ultrasound, enhanced ectopic bone formation was evident in 100% of mice treated with GAM and ultrasound, with a 5.7-fold increase compared to passive GAM (no ultrasound) and a 16.44-fold increase compared to standard sonoporation (injection of DNA and microbubbles followed by ultrasound, no GAM/exogenous cells). An interesting finding from this study was that only 6.5 µg of DNA were needed to elicit this response; a previous study from 2014, the first to evaluate sonoporation with BMP2/7 co-expression plasmids in a bone defect model, found it necessary to use 100 µg of DNA in multiple consecutive injections, along with a much higher ultrasound energy density [bib_ref] Sonoporation Increases Therapeutic Efficacy of Inducible and Constitutive BMP2/7 In Vivo Gene..., Feichtinger [/bib_ref]. In this study by Feichtinger et al., results included irregular ectopic bone structure, multiple centers of ossification, and skin burning at the exit site of the ultrasound when using a 4 W/cm 2 protocol. A possible advantage of using a GAM is that while formulated as a liquid, the fibrin gelling time can be adjusted to occur directly after injection, and this may minimize off-target bone generation.
The study by Nomikou et al. emphasized the importance of specific "responder" cells at the target site. This point was further demonstrated by a study in Yucatan minipigs with a critical-sized bone fracture in the tibia, where a collagen scaffold was then implanted to facilitate the recruitment of endogenous mesenchymal stem cells (MSCs) into the fracture site [bib_ref] In situ bone tissue engineering via ultrasound-mediated gene delivery to endogenous progenitor..., Bez [/bib_ref]. Two weeks after the defect was created, transcutaneous ultrasoundmediated delivery of hBMP-6 plasmid led to complete radiographic and functional fracture healing in all animals at 6 weeks post-sonoporation. In control animals, nonunion was evident. This study, the only one that has reported on the use of sonoporation for tissue regeneration in large animals thus far, suggested that the delayed administration of BMP-6 may have allowed for sufficient endogenous progenitor migration and retention in the scaffold at the target site prior to treatment. This approach has been previously suggested in a study that delayed the administration of a BMP-2-encoding adenoviral vector and found improved bone formation in a critical-sized femoral fracture in rats [bib_ref] Delayed administration of adenoviral BMP-2 vector improves the formation of bone in..., Betz [/bib_ref]. Compared to the sonoporation group, similar results were found in animals treated with a bone autograft, the current standard of care, suggesting that the sonoporation method used was as efficient; the authors also noted that the bone autografts used in this experiment might be far superior to autografts typically available in the clinical setting [bib_ref] In situ bone tissue engineering via ultrasound-mediated gene delivery to endogenous progenitor..., Bez [/bib_ref].
Targeting endogenous progenitor/stem cells using sonoporation was also demonstrated in skeletal soft tissues. Delalande et al. had previously established optimized parameters for effective gene expression to tendons [bib_ref] Ultrasound-assisted microbubbles gene transfer in tendons for gene therapy, Delalande [/bib_ref]. In a separate study, an anterior cruciate ligament (ACL) reconstruction procedure was conducted in Yucatan mini pigs. Allogeneic tendon grafts were secured in femoral and tibial bone tunnels, and a collagen scaffold was implanted around the grafts to attract endogenous progenitor/stem cells [bib_ref] Ultrasound-Mediated Gene Delivery Enhances Tendon Allograft Integration in Mini-Pig Ligament Reconstruction, Bez [/bib_ref]. Two weeks after the tendon graft and scaffold implantation, transcutaneous ultrasound was used to deliver a BMP-6 gene. Results showed that, in addition to a 15-fold increase in the expression of BMP6 in animals treated with ultrasound, osteointegration of the tendon grafts was significantly enhanced. Animals treated with sonoporation exhibited twice the bone volume as the control animals, and the researchers noted tissue continuity and a lack of ectopic bone formation. The absence of mononucleated cells in the grafts post-mortem suggested that sonoporation did not invoke an inflammatory reaction. [fig_ref] Table 3: The use of sonoporation for skeletal tissue regeneration [/fig_ref] summarizes the studies that have used sonoporation for skeletal tissue regeneration thus far. Despite promising results, improvements in transfection efficiency are still desired. Studies have found that adjustments to ultrasound parameters can increase gene expression in the bone defect model for up to 21 days [bib_ref] Multiparameter evaluation of in vivo gene delivery using ultrasound-guided, microbubble-enhanced sonoporation, Shapiro [/bib_ref]. However, sonoporation is especially challenging in sites involving bone and metal implants, such as those used in the fixation of fractures, as they are highly reflective of ultrasound waves. Hence, efficient transfection may require longer ultrasound times [bib_ref] Ultrasound-assisted microbubbles gene transfer in tendons for gene therapy, Delalande [/bib_ref] , but this may lead to adverse side effects. Sonoporation applied with intramuscular injection of rhBMP-9 plasmid and lipid-stabilized microbubbles resulted in ectopic bone formation [bib_ref] Ultrasound-based nonviral gene delivery induces bone formation in vivo, Sheyn [/bib_ref] Rich-Mar Sonitron 2000
Repeated sonoporation with BMP-2 plasmid significantly increased osteoinduction compared to one treatment session [bib_ref] Osteoinduction by microbubble-enhanced transcutaneous sonoporation of human bone morphogenetic protein-2, Osawa [/bib_ref] Sonidel SP100
Using 4 W/cm 2 sonoporation and constitutive BMP2/7 co-expression plasmid significantly increased ectopic bone formation, but with variable morphology and irregular shape [bib_ref] Sonoporation Increases Therapeutic Efficacy of Inducible and Constitutive BMP2/7 In Vivo Gene..., Feichtinger [/bib_ref] Sonidel SP100
Use of a GAM and BMP2/7 co-expression plasmid significantly enhanced ectopic bone formation compared to standard sonoporation [bib_ref] Ultrasound-responsive gene-activated matrices for osteogenic gene therapy using matrix-assisted sonoporation, Nomikou [/bib_ref] Femur Defect Rat Sonidel SP100 1
Use of a BMP2/7 co-expression plasmid resulted in fracture union in 33% of rats, compared to the 0% union rate in the control group, although this result was not statistically significant [bib_ref] Sonoporation Increases Therapeutic Efficacy of Inducible and Constitutive BMP2/7 In Vivo Gene..., Feichtinger [/bib_ref] Tibia
## Treatment of myocardial ischemia with sonoporation
Cardiac wound healing in mammals is severely limited due to the development of scar tissue and the necrosis that results due to the loss of blood supply [bib_ref] Post-infarct remodelling: Contribution of wound healing and inflammation, Frantz [/bib_ref]. To try to address this lack of self-regeneration, a preliminary study from 2012 used ultrasound and microbubbles to deliver the thymosin beta 4 (TB4) gene under a piggyBac transposon plasmid to normal rat hearts [bib_ref] Stimulation of adult resident cardiac progenitor cells by durable myocardial expression of..., Chen [/bib_ref]. TB4 was found to stimulate angiogenesis and arteriogenesis and promote the proliferation and differentiation of resident WT1-positive adult cardiac progenitor cells into three intact cardiac cell lineages: vascular endothelial cells, coronary artery smooth muscle cells, and cardiac muscle cells. This study provided support for the use of sonoporation for cardiac regeneration, whereas the first study to demonstrate this was published in 2009 [bib_ref] Ultrasound-Targeted Gene Delivery Induces Angiogenesis After a Myocardial Infarction in Mice, Fujii [/bib_ref]. In this study from 2009, lipid microbubbles and plasmid DNA were injected intravenously into mice 7 days after coronary artery ligation, which is used to model a myocardial infarction (MI). The plasmid injected was encoded for either vascular endothelial growth factor (VEGF) or stem cell factor (SCF). Results from this study showed that injection of either plasmid yielded greater capillary and arteriolar density, myocardial perfusion, and enhanced cardiac function compared to the control group, which received empty plasmids. A follow-up study was conducted by the same authors to determine the effect of multiple treatments in the MI rat model [bib_ref] Repeated and targeted transfer of angiogenic plasmids into the infarcted rat heart..., Fujii [/bib_ref]. They found that multiple injections of SCF and stromal cell-derived factor-1α (SDF-1α) resulted in the greatest increase in vascular densities compared to the control, although all sonoporation recipients did exhibit an increase in vascular density and a smaller infarct region. Myocardial perfusion and ventricular function also improved progressively with the number of treatments.
Soon after, the use of cationic microbubbles was compared to commercially available lipid microbubbles. This comparison was assessed based on the ability to deliver the therapeutic AKT gene, a serine/threonine-protein kinase, to ischemic rat myocardium via intravenous injection [bib_ref] The use of cationic microbubbles to improve ultrasound-targeted gene delivery to the..., Sun [/bib_ref]. The authors found that the cationic microbubbles bound 70% more plasmid DNA, and they attributed this to the high zeta potential of cationic microbubbles. In an ischemia/reperfusion (I/R) rat model, the authors demonstrated that AKT transfection reduced infarct size, increased infarct thickness, and reduced apoptosis. It was also found that ejection fraction was significantly improved and that the densities of the capillaries and arterioles within the border region of the infarct were significantly increased compared to the control animals treated with a vector control or lipid microbubbles. The study concluded that cationic microbubble-based delivery of the AKT gene produced the greatest increase in ventricular function and myocardial perfusion.
Unlike previous studies, which injected a plasmid, a more recent study aimed to increase the delivery of antagomir to the myocardium of both healthy control mice and in an I/R injury mouse model [bib_ref] Increased local delivery of antagomir therapeutics to the rodent myocardium using ultrasound..., Kwekkeboom [/bib_ref]. Antagomir is a microRNA (miRNA)-inhibitor, but it has low myocardial specificity, and thus, cardiac treatment with antagomir requires high doses, which can result in adverse side effects. The researchers found that ultrasound and cationic microbubbles significantly increased local antagomir delivery to the non-ischemic heart; they also noted only modest side effects, including neutrophil invasion, but did not observe an increase in apoptosis. The findings from this study also suggest that antagomir enters cardiomyocytes within 30 min post-treatment and remains there for at least 48 h. After I/R injury, antagomir readily enters the infarcted zone, but the results did not show any additional regenerative benefits when using the ultrasound. Interestingly, this study demonstrated that the extent and location of antagomir delivery were dependent on the ultrasound frequency and mode. While delivery occurred mostly to the anterior wall of the heart, a higher frequency led to more restrictive delivery to the anterior wall, while a lower frequency enabled delivery to more parts of the heart. In support of the proposed safety of sonoporation, this study found that any damage to the heart was local and temporal and that treatment did not cause damage to the cardiomyocytes themselves.
The use of miRNA-inhibitors (antimiR) and sonoporation has also been proposed to prevent the destruction of heart tissue. Researchers found that multiple treatments with cationic microbubbles and antimiR-23a in a phenylephrine-induced cardiac hypertrophy mouse model resulted in a 41% decrease in cardiac miR-23a levels, a decrease in the mass of the left ventricle, and a higher fractional shortening [bib_ref] Ultrasound and Microbubble-targeted Delivery of a microRNA Inhibitor to the Heart Suppresses..., Kopechek [/bib_ref]. Cardiac levels of hypertrophic mRNAs (ANP and MYH7) also decreased, but this difference did not reach statistical significance. This suggests that ultrasound significantly reduces the antimiR dose needed for therapeutic efficacy by a factor of over 200-fold. However, this does not address whether treatment can cause a regression in established or chronic cardiac hypertrophy. In addition, the researchers saw a loss of protection against left ventricular hypertrophy by one-week post-ultrasound treatment, with ongoing phenylephrine infusions, but did note increased protection against systolic dysfunction during ultrasound treatments.
Similar to the use of sonoporation for skeletal tissue regeneration, the optimal ultrasound parameters and injection method for cardiac transfection still need to be confirmed. For example, a study in 2015 aimed to transfect microRNA-21 (miR-21) in healthy swine hearts and wanted to compare the efficacy of intravenous injection to that of injection directly into the myocardium [bib_ref] Ultrasound-Targeted Microbubble Destruction Enhances Gene Expression of microRNA-21 in Swine Heart via..., Liu [/bib_ref]. The authors found that an ultrasound intensity of 2 W/cm 2 and a 50% duty ratio for 20 min was best and did not cause any injury to the myocardium. Using these parameters, they found an increase in gene expression in the myocardium, regardless of the miR-21 delivery route, but they did note a slight increase in transfection efficiency with intracoronary injection; it is important to note that this increase in transfection efficiency was observed even when the dose for the intracoronary injection was half that of the intravenous injection. This study also suggested that preconditioningregulated miR-21 could protect the heart against injury via anti-apoptosis through its target PDCD4.
In addition, in an effort to increase the efficiency of gene delivery for cardiac regeneration, a recent study explored the use of sonoporation for the delivery of therapeutic genes to an MI rat model, using microbubbles conjugated to adenoviruses encoding for the Ca 2+ ATPase 2a (SERCA2a) and connexin 43 (Cx43) genes [bib_ref] Pre-transplantation of Bone Marrow Mesenchymal Stem Cells Amplifies the Therapeutic Effect of..., Wang [/bib_ref]. This study showed that animals that received both genes had the best cardiac contractile function and electrical stability compared to controls, although there was evidence of a change in the infarct size. Interestingly, the therapeutic efficacy was further increased when bone marrow MSCs were injected at the infarct site and border zones 4 weeks prior to sonoporation. The authors did not determine whether the injected cells were subsequently transfected during the sonoporation process, but they did hypothesize, based on previous studies, that the stem cells helped to maintain an important population of cardiomyocytes that were targeted for transfection. This study further supports the importance of targeting the right responder cells at the site that requires regeneration and proposes the potential benefit of using sonoporation in conjunction with other gene delivery systems to maximize the resulting therapeutic effects. [fig_ref] Table 4: The use of sonoporation for cardiac tissue regeneration [/fig_ref] summarizes the studies that used sonoporation for cardiac tissue regeneration, but it is evident that additional research is needed.
## Treatment of peripheral ischemia with sonoporation
Like the biological limitations of resolving a myocardial infarction, peripheral artery disease also cannot be resolved through self-regeneration. In a rabbit peripheral ischemia model, hepatocyte growth factor (HGF) plasmid and lipid microbubbles were injected locally into the pretibial muscle, followed by ultrasound application [bib_ref] Development of safe and efficient novel nonviral gene transfer using ultrasound: Enhancement..., Taniyama [/bib_ref]. Five weeks after transfection, the angiographic score and capillary density of animals treated with ultrasound significantly increased compared to those only injected with the HGF plasmid.
This was accompanied by a significant increase in blood flow and in the blood pressure ratio. This study provided evidence that sonoporation could be a safe and useful gene-based therapy to treat peripheral artery disease.
In a rat model of severe chronic hindlimb ischemia, a study showed that VEGF-165 plasmid, infused intravenously over 10 min, and ultrasound application resulted in a significant improvement in microvascular blood flow and in an increase in vessel density [bib_ref] Therapeutic Arteriogenesis by Ultrasound-Mediated VEGF 165 Plasmid Gene Delivery to Chronically Ischemic..., Leong-Poi [/bib_ref]. Improvement in tissue perfusion was attributed to increases in noncapillary blood volume (arteriogenesis), with perfusion peaking at 14 days post-treatment, followed by a partial regression of neovascularization at 6 weeks. Transfection was localized primarily to the vascular endothelium of arterioles. Later, a similar study design compared the efficacy of treatment with different sites of injection, intravenous (IV) and intramuscular (IM) [bib_ref] Comparison of Gene Delivery Techniques for Therapeutic Angiogenesis: Ultrasound-Mediated Destruction of Carrier..., Kobulnik [/bib_ref]. They found that both IM and IV delivery produced significant increases in microvascular blood volume and microvascular blood flow, but microvascular blood flow was greater in IV-treated animals, even 8 weeks post-treatment. Interestingly, VEGF165/GFP mRNA expression was significantly greater in IM-treated animals. Intravenous delivery resulted in directed vascular transfection over a wider distribution, which may account for the greater degree of angiogenesis in this group.
More recently, a study was designed to compare temporally separated VEGF and angiopoetin-1 (Ang-1) delivery to concomitant delivery and single VEGF delivery for therapeutic angiogenesis in a chronic unilateral hindlimb ischemia rat model [bib_ref] Sustained Improvement in Perfusion and Flow Reserve After Temporally Separated Delivery of..., Smith [/bib_ref]. Using cationic microbubbles, the authors found that VEGF delivery improved blood flow and vessel density, but flow reserve remained low, and supporting cell coverage in the new vessels was poor. VEGF/Ang-1 co-delivery marginally increased blood flow and vessel density, but the authors did note that co-delivery improved flow reserve and supporting cell coverage. Temporally separated VEGF and Ang-1 delivery, with VEGF delivered at 2 weeks after ligation and Ang-1 delivered at 4 weeks post-ligation, resulted in increased blood flow and vessel density and increased and sustained the flow reserve, with improved pericyte coverage at 8 weeks.
Lastly, Cao et al. published a sonoporation study in 2015, using miR-126-3p and cationic microbubbles in rats following chronic left femoral artery ligation [bib_ref] Therapeutic Angiogenesis by Ultrasound-Mediated MicroRNA-126-3p Delivery, Cao [/bib_ref]. Treatment of the chronic ischemic hindlimb muscle resulted in improved perfusion and vessel density, enhanced arteriolar formation, pericyte coverage, and phosphorylated Tie2 levels and did not affect miR-126-5p or delta-like 1 homolog levels. The authors speculated that the observed biological effect was a result of suppressing sprouty-related protein-1 (SPRED1) and phosphatidylinositol-3-kinase regulatory subunit 2 (PIK3R2) and enhancing VEGF and Ang-1 signaling. This study also showed that the use of cationic microbubbles stabilized and extended the circulatory time in vivo, finding that conjugating the microbubbles and miRNA prior to injection led to prolonged circulatory time compared to that of unbound miRNA. Overall, this study showed that treatment with miR-126-3p resulted in significant improvements in microvascular perfusion, with minimal to no uptake in remote organs. Animals that underwent repeated injection and ultrasound treatment exhibited an even greater angiogenic response. [fig_ref] Table 5: The use of sonoporation for ischemia in peripheral tissue [/fig_ref] summarizes the studies that have explored the use of sonoporation as a therapy for peripheral ischemia thus far. Angiographic score and capillary density of animals treated with ultrasound and HGF plasmid was significantly greater than the control, resulting in a significant increase in blood flow and blood pressure ratio [bib_ref] Development of safe and efficient novel nonviral gene transfer using ultrasound: Enhancement..., Taniyama [/bib_ref] Rat Philips Sonos 5500; S3 transducer 1.3
Infusion of VEGF-165 plasmid resulted in significant improvement in microvascular blood flow and increased vessel density, with transfection localized predominantly to the vascular endothelium of arterioles [bib_ref] Therapeutic Arteriogenesis by Ultrasound-Mediated VEGF 165 Plasmid Gene Delivery to Chronically Ischemic..., Leong-Poi [/bib_ref] Both IM and IV delivery of VEGF-165 plasmid produced significant increases in microvascular blood volume and blood flow, but microvascular blood flow was greater in IV-treated animals [bib_ref] Comparison of Gene Delivery Techniques for Therapeutic Angiogenesis: Ultrasound-Mediated Destruction of Carrier..., Kobulnik [/bib_ref] Temporally separated VEGF and Ang-1 plasmid delivery resulted in increased blood flow, vessel density, and sustained an increase in flow reserve [bib_ref] Sustained Improvement in Perfusion and Flow Reserve After Temporally Separated Delivery of..., Smith [/bib_ref] Treatment with miR-126-3p resulted in significant improvements in microvascular perfusion, and repeated treatment exhibited an even greater angiogenic response [bib_ref] Therapeutic Angiogenesis by Ultrasound-Mediated MicroRNA-126-3p Delivery, Cao [/bib_ref]
## Sonoporation for pancreatic islet regeneration
Pancreatic islets contain multiple cell types, including beta (β) cells that produce insulin, and are therefore a treatment target in the effort to reverse diabetes mellitus. The first study that demonstrated the ability of ultrasound-targeted gene therapy to regenerate pancreatic islets, thus leading to recovery from diabetes, without the use of a viral vector, was published in 2010 [bib_ref] Regeneration of pancreatic islets in vivo by ultrasound-targeted gene therapy, Chen [/bib_ref]. Chen et al. utilized lipid-stabilized microbubbles with a modified rat insulin promoter (RIP3.1) for greater targeting to β-cells, in a streptozotocin (STZ)-induced diabetic rat model. Several genes were used, but while PAX4, Nkx2.2, Nkx6.1, Ngn3, and Mafa produced alpha-cell hyperplasia, there was no significant improvement in the β-cell mass or in blood glucose levels 30 days post-sonoporation. In contrast, injection of RIP3.1-NeuroD1 promoted islet regeneration from surviving β-cells, resulting in the normalization of glucose, insulin, and C-peptide levels at 30 days post-treatment. In a longer-term experiment, 4 out of 6 rats had a return of diabetes by 90 days, which was accompanied by β-cell apoptosis. However, for rats pretreated with SP600125, a JNK inhibitor, β-cell apoptosis was restricted, and researchers noted an extension in the duration of islet regeneration and normoglycemia to 90 days, suggesting that an immunosuppressive regimen for islet protection may need to be established going forward.
A similar study from this group delivered islet transcription factor genes using a piggyBac transposon gene delivery system for long-term transgene expression of Nkx2.2 in the pancreas of an adult diabetic rat model [bib_ref] Ectopic transgenic expression of NKX2.2 induces differentiation of adult pancreatic progenitors and..., Chen [/bib_ref]. Results showed that the Nkx2.2 gene induced robust proliferation and differentiation of adult pancreatic progenitors. Using high-resolution confocal images, the authors were able to show how one differentiated pancreatic progenitor cell developed into islet-like clusters, and then into mature islets with normal morphology. This pancreatic islet regeneration process enabled a reversal in STZ-induced diabetes for 3 months.
Previous work has shown that gene therapy with cyclin D2/CDK4/GLP-1 plasmids targeted to the pancreas of STZ-treated rats could force cell cycle re-entry of residual G 0 -phase islet cells into the G 1 /S phase in order to regenerate β cells [bib_ref] Transient overexpression of cyclin D2/CDK4/GLP1 genes induces proliferation and differentiation of adult..., Chen [/bib_ref]. A single treatment of sonoporation induced β-cell regeneration with reversal of diabetes for 6 months, without evidence of toxicity or the activation of oncogenes. Cyclin D2/CDK4/GLP-1 gene delivery initiated robust proliferation of the adult pancreatic progenitor cells that exist within islets. A similar study used the ANGPTL8 gene, delivered to the pancreas, liver, and skeletal muscle of normal adult rats [bib_ref] In vivo targeted delivery of ANGPTL8 gene for beta cell regeneration in..., Chen [/bib_ref]. ANGPTL8 was detected in circulation 1-month post-treatment. Sonoporation with ANGPTL8 significantly alleviated but did not totally reverse STZ-induced diabetes in this rat model. The largest improvement, however, was seen when ANGPTL8 was targeted to the pancreas. Pancreas-targeted treatment promoted the proliferation of adult and aged β cells, expanded the β cell mass, improved glucose tolerance, and increased the fasting blood insulin levels without causing severe hypertriacylglycerolaemia. The authors suggested that the lack of disease reversal may be due to the fact that the treated animals did not have enough β cells to undergo replication in sufficient numbers to actually reverse the disease.
A more recent study hypothesized that gene therapy with a plasmid cDNA cocktail of BMP7/PRDM16/PPARGC1A, injected into the skeletal muscle of obese Zucker diabetic fatty rats, would produce a brown adipose tissue phenotype with UCP-1 overexpression [bib_ref] Engineering brown fat into skeletal muscle using ultrasound-targeted microbubble destruction gene delivery..., Bastarrachea [/bib_ref]. This proof-of-concept project used lipid-stabilized microbubbles injected intravenously. Treatment produced a reliable pattern of enhancing skeletal muscle overexpression of the UCP-1 gene, indicating that this plasmid DNA construct may be capable of reprogramming adult skeletal muscle tissue into brown adipose cells in vivo. [fig_ref] Table 6: The use of sonoporation as a therapy for diabetes [/fig_ref] provides an overview of the studies that have demonstrated the ability of sonoporation to induce the regeneration of pancreatic islet cells, but this study highlights the additional ways in which sonoporation may be used to ameliorate the diabetic condition, supporting the need for additional research in this area. RIP3.1-NeuroD1 plasmid promoted islet regeneration from surviving beta-cells, with normalization of glucose, insulin, and C-peptide levels up to 30 days, but pretreating with SP600125 could extend the duration of islet regeneration and normoglycemia to 90 days [bib_ref] Regeneration of pancreatic islets in vivo by ultrasound-targeted gene therapy, Chen [/bib_ref] Injection of the Nkx2.2 gene induced robust proliferation and differentiation of adult pancreatic progenitors, curing STZ-induced diabetes for 3 months [bib_ref] Ectopic transgenic expression of NKX2.2 induces differentiation of adult pancreatic progenitors and..., Chen [/bib_ref] A single sonoporation treatment with cyclin D2/CDK4/GLP-1 plasmids induced β-cell regeneration with reversal of diabetes for 6 months without evidence of toxicity or activation of oncogenes [bib_ref] Transient overexpression of cyclin D2/CDK4/GLP1 genes induces proliferation and differentiation of adult..., Chen [/bib_ref] ANGPTL8 gene targeted to the pancreas significantly alleviated but did not totally reverse STZ-induced diabetes, but treatment did promote the proliferation of adult and aged beta cells, expanding the beta-cell mass and improving glucose tolerance [bib_ref] In vivo targeted delivery of ANGPTL8 gene for beta cell regeneration in..., Chen [/bib_ref] 3.
## Other applications of sonoporation for tissue regeneration
In addition to the studies detailed above, sonoporation has also been tested in other models of tissue regeneration. The use of sonoporation for dental conditions is desirable because current gene delivery methods exhibit low transference and efficiency in the pulp space. In 2002, Nakashima et al. aimed to use gene therapy to induce reparative dentin formation for potential pulp capping since the conventional method, using calcium hydroxide, only induces a small amount of reparative dentin [bib_ref] Induction of Reparative Dentin Formation by Ultrasound-Mediated Gene Delivery of Growth/Differentiation Factor..., Nakashima [/bib_ref]. This group of researchers had previously optimized the transference of a growth/differentiation factor 11 (Gdf11)/BMP11 plasmid into dental pulp stem cells by in vivo electroporation but also noted an accumula-tion of erythrocytes in a plasma clot adjacent to the electrode, potentially caused by thermal effects. As a result of these findings, the authors hoped to eliminate the adverse effects by utilizing sonoporation instead [bib_ref] Induction of dental pulp stem cell differentiation into odontoblasts by electroporation-mediated gene..., Nakashima [/bib_ref]. While therapeutic reparative dentin formation can be achieved with BMPs [bib_ref] Induction of Dentin Formation on Canine Amputated Pulp by Recombinant Human Bone..., Nakashima [/bib_ref] , the half-life of the morphogen is a limiting factor, and the BMPs must reach the pulp tissue stem cells in order to be effective. Sonoporation of Gdf11 stimulated a large amount of reparative dentin formation in the amputated dental pulp of canine teeth in vivo. This was a promising result, suggesting that sonoporation may be useful in endodontic dental treatment, although no in vivo dental regeneration studies have been conducted since. In 2014, Sugano et al. were able to optimize the intensity and exposure time of sonoporation for treating periodontitis in rats, although this study was not designed to demonstrate tissue regeneration, and thus, did not use genes relevant for regeneration [bib_ref] Gene delivery to periodontal tissue using Bubble liposomes and ultrasound, Sugano [/bib_ref].
Two recent studies have investigated how the principles of sonoporation can be used to increase hair follicle growth [bib_ref] Minoxidil-Coated Lysozyme-Shelled Microbubbes Combined with Ultrasound for the Enhancement of Hair Follicle..., Liao [/bib_ref] [bib_ref] Ultrasound-activated particles as CRISPR/Cas9 delivery system for androgenic alopecia therapy, Ryu [/bib_ref]. Liao et al. focused on the potential of inhibiting bacteria and allergies on the scalp with the use of lysozyme-shelled microbubbles, finding that ultrasound application significantly enhanced hair growth rates in mice [bib_ref] Minoxidil-Coated Lysozyme-Shelled Microbubbes Combined with Ultrasound for the Enhancement of Hair Follicle..., Liao [/bib_ref]. Ryu et al. designed a microbubble-nanoliposomal particle to act as a Cas9/sgRNA riboprotein complex carrier and found that the protein constructs were successfully transferred into dermal papilla cells, effectively treating the mice with androgenic alopecia [bib_ref] Ultrasound-activated particles as CRISPR/Cas9 delivery system for androgenic alopecia therapy, Ryu [/bib_ref]. The results of this study demonstrated that the external application of ultrasound allowed the treatment to diffuse deeper and that the riboprotein complex carrier experienced a high efficiency of recognition and gene editing, with an on-target effect of about 70%. The authors of this study encouraged future research on how this method could be applied to the treatment of skin carcinoma and melanoma.
Sonoporation has also been used to reverse the innate reparative processes of the body, such as the creation of fibrotic tissue. Cirrhosis, a chronic liver disease, occurs due to the progressive deterioration of liver function and results in fibrosis, which may lead to liver failure. A study found that intravenous delivery of the hepatocyte growth factor (HGF) gene and application of ultrasound markedly attenuated fibrosis in rats and resulted in a significant decrease in the serum levels of alanine transaminase (ALT) and aspartate transaminase (AST), and the expression of collagen I, collagen II, and α-SMA proteins [bib_ref] Attenuation of hepatic fibrosis through ultrasound-microbubblemediated HGF gene transfer in rats, Jiang [/bib_ref]. Soon after this study was published, another examined the antifibrotic effect of an artificial microRNA designed to target a connective tissue growth factor (CTGF). Conjugated with cationic microbubbles, using a biotin-avidin system, this study showed that repeated delivery of artificial miRNAs is safe and convenient, and the results demonstrated that the progression of hepatic fibrosis was effectively ameliorated, and cirrhosis was successfully prevented by downregulating CTGF expression [bib_ref] Inhibition of hepatic fibrosis with artificial microRNA using ultrasound and cationic liposome-bearing..., Yang [/bib_ref]. Despite effective delivery to the liver, however, liver injury was not reversed. This finding suggests that the induction of healthy tissue growth may not always be sufficient to reverse a chronic condition and that efforts to address innate scarring processes, for example, may also need to be employed.
## Considerations for clinical translation
Currently, sonoporation is only cited in three ongoing clinical trials. Although up to 115 studies reference the use of ultrasound and microbubbles, most studies focus on the imaging and diagnostic applications or are using ultrasound-induced cavitation for thrombolysis. However, of the three ongoing clinical trials using sonoporation, none utilize sonoporation for gene delivery nor for tissue regeneration. The current clinical utilization of sonoporation is to enhance the targeted delivery of chemotherapeutic agents [bib_ref] Ultrasound and microbubbles to beat barriers in tumors: Improving delivery of nanomedicine, Snipstad [/bib_ref]. The preliminary results from these trials, including one in which sonoporation is used to disrupt the blood-brain barrier in patients with recurrent glioblastoma [bib_ref] Safety and Feasibility of Repeated and Transient Blood-Brain Barrier Disruption by Pulsed..., Idbaih [/bib_ref] , demonstrate that systemic delivery of microbubbles and repeated ultrasound exposure is a safe practice for humans.
That said, there are various factors which need to be addressed further before this application of sonoporation can be translated to the clinical setting. These factors primarily pertain to the specifics of the microbubble and plasmid injection. The current body of research has not been able to conclusively identify the best type of microbubbles to use, although it appears that cationic microbubbles may increase transfection compared to lipid-coated microbubbles [bib_ref] Ultrasound-mediated gene transfer (sonoporation) in fibrin-based matrices: Potential for use in tissue..., Nomikou [/bib_ref] [bib_ref] The use of cationic microbubbles to improve ultrasound-targeted gene delivery to the..., Sun [/bib_ref] , nor have researchers been able to establish the best timeline for gene delivery [bib_ref] Sustained Improvement in Perfusion and Flow Reserve After Temporally Separated Delivery of..., Smith [/bib_ref]. While it appears that there are benefits to the co-delivery of genes [bib_ref] Ultrasound-responsive gene-activated matrices for osteogenic gene therapy using matrix-assisted sonoporation, Nomikou [/bib_ref] [bib_ref] Sonoporation Increases Therapeutic Efficacy of Inducible and Constitutive BMP2/7 In Vivo Gene..., Feichtinger [/bib_ref] [bib_ref] Ultrasound-Targeted Microbubble Destruction-Mediated Co-Delivery of Cxcl12 (Sdf-1alpha) and Bmp2 Genes for Myocardial..., Yang [/bib_ref] , it will be important to determine the best ratio of the genes and whether, indeed, the genes should be delivered at the same time or if they should be delivered at separate times [bib_ref] Sustained Improvement in Perfusion and Flow Reserve After Temporally Separated Delivery of..., Smith [/bib_ref]. In addition, especially in cases when a scaffold is utilized in the target site, while it has been found that delayed administration of plasmid DNA allows for sufficient endogenous progenitor cell migration [bib_ref] In situ bone tissue engineering via ultrasound-mediated gene delivery to endogenous progenitor..., Bez [/bib_ref] [bib_ref] Ultrasound-Mediated Gene Delivery Enhances Tendon Allograft Integration in Mini-Pig Ligament Reconstruction, Bez [/bib_ref] [bib_ref] Delayed administration of adenoviral BMP-2 vector improves the formation of bone in..., Betz [/bib_ref] [bib_ref] Ultrasound-assisted microbubbles gene transfer in tendons for gene therapy, Delalande [/bib_ref] , it will be necessary to determine the most optimal delay in sonoporation.
Clarification on when the ultrasound should be applied, with respect to the injection, is also needed. Some studies have utilized continuous infusion during ultrasound application [bib_ref] The use of cationic microbubbles to improve ultrasound-targeted gene delivery to the..., Sun [/bib_ref] , while others have completely infused the plasmid and microbubbles and then applied the ultrasound [bib_ref] Ultrasound-Targeted Gene Delivery Induces Angiogenesis After a Myocardial Infarction in Mice, Fujii [/bib_ref]. It may be the case that the ultrasound should be applied during infusion and then for a set time after the infusion has been completed to ensure that all microbubbles still in circulation have been destroyed [bib_ref] Therapeutic Arteriogenesis by Ultrasound-Mediated VEGF 165 Plasmid Gene Delivery to Chronically Ischemic..., Leong-Poi [/bib_ref]. In addition, while repeated treatments have been found to be safe and may result in greater regeneration [bib_ref] Osteoinduction by microbubble-enhanced transcutaneous sonoporation of human bone morphogenetic protein-2, Osawa [/bib_ref] [bib_ref] Repeated and targeted transfer of angiogenic plasmids into the infarcted rat heart..., Fujii [/bib_ref] , the timeline of treatment must be assessed further, as there is still the risk of muscle damage and inflammatory reaction with each intramuscular injection [bib_ref] Inflammatory responses following direct injection of plasmid DNA into skeletal muscle, Mcmahon [/bib_ref].
Additionally, the site of injection is important to study further; while the majority of studies utilize intravenous injection, it will be important to deduce how direct injection impacts transfection efficiency, as one study found that intracoronary injection of a halfdose resulted in slightly higher transfection efficiency than intravenous injection [bib_ref] Ultrasound-Targeted Microbubble Destruction Enhances Gene Expression of microRNA-21 in Swine Heart via..., Liu [/bib_ref]. Despite this, intravenous injection appears to be a safe practice and may present the least burdensome injection method, which would further support the adoption of this method in the clinical setting.
The location of where the ultrasound is applied may also need to be studied further. Transcutaneous sonoporation has been found to successfully induce osteoinduction [bib_ref] Osteoinduction by microbubble-enhanced transcutaneous sonoporation of human bone morphogenetic protein-2, Osawa [/bib_ref] but may be limited in its clinical use for fractures located in deeper tissues, such as in the case of hip fractures [bib_ref] Low-Intensity Pulsed Ultrasound Stimulation for Bone Fractures Healing: A Review, Be [/bib_ref]. The concept of implantable sonoporation devices has not yet been heavily studied for tissue regeneration, but cases that have used sonoporation for blood-brain barrier disruption in the treatment of brain disorders have found it to be beneficial. Particularly, implantation enables researchers to avoid the large ultrasound wave attenuation caused by the skull when ultrasound is applied externally [bib_ref] Safety and Feasibility of Repeated and Transient Blood-Brain Barrier Disruption by Pulsed..., Idbaih [/bib_ref].
While sonoporation has been found to have minimal to no off-target delivery, studies have demonstrated that the frequency and mode of the ultrasound may alter the extent of delivery to certain tissues. This was seen in the study by where delivery primarily occurred to the anterior wall of the heart unless a lower frequency was used [bib_ref] Increased local delivery of antagomir therapeutics to the rodent myocardium using ultrasound..., Kwekkeboom [/bib_ref]. Despite the current collection of pre-clinical studies on the induction of tissue regeneration by sonoporation, there is a clear need for greater guidance on how to optimize the therapeutic benefits, although this may likely differ depending on the targeted organ system.
# Conclusions
The application of ultrasound as a method to increase gene delivery is advantageous because ultrasound has already been integrated into clinical practices, and it allows for the visual monitoring of the transfection process when microbubbles are co-injected. While the in vivo studies discussed above present promising results for the use of sonoporation for tissue regeneration, the lack of current clinical trials may allude to the existence of additional hurdles that need to be addressed in pre-clinical, regeneration-focused research. Despite this, the clinical trials currently utilizing sonoporation show that it is a safe practice in humans, which should lend support to the development of clinical protocols specifically targeting tissue regeneration.
In addition to the limitations addressed above, there are also concerns regarding the scalability of sonoporation, as most of the pre-clinical experiments only utilized small animal models. A recent study on acousticfluidic sonoporation appears to present encouraging results for increasing the scalability of intracellular delivery, achieving a throughput of 200,000 cells per minute in a single channel [bib_ref] Acoustofluidic sonoporation for gene delivery to human hematopoietic stem and progenitor cells, Belling [/bib_ref] , although large animal studies will be needed to further evaluate this technique.
A greater understanding of how sonoporation impacts other biochemical processes may be necessary prior to clinical implementation. For example, it has been suggested that more research is needed to characterize the effect that sonoporation has on extracellular vesicles (EVs), as the release of EVs seems to be part of the action to reseal the disrupted plasma membrane, with one study reporting a significant increase in EV release between 2 and 4 h after sonoporation [bib_ref] Microbubbles-Assisted Ultrasound Triggers the Release of Extracellular Vesicles, Yuana [/bib_ref].
A few studies have proposed that combining sonoporation with other transfection strategies may have the potential to increase transfection efficiency and the therapeutic benefits of sonoporation. The coordinated use of sonoporation with other gene delivery systems, including viral vector injection [bib_ref] Cavitation-enhanced delivery of a replicating oncolytic adenovirus to tumors using focused ultrasound, Bazan-Peregrino [/bib_ref] or magneto-sonoporation [bib_ref] Rapid Magneto-Sonoporation of Adipose-Derived Cells, Filippi [/bib_ref] , may further enhance the regenerative potential of sonoporation [bib_ref] Ultrasound-Targeted Microbubble Destruction-Mediated Co-Delivery of Cxcl12 (Sdf-1alpha) and Bmp2 Genes for Myocardial..., Yang [/bib_ref] , as suggested in a recent study [bib_ref] Pre-transplantation of Bone Marrow Mesenchymal Stem Cells Amplifies the Therapeutic Effect of..., Wang [/bib_ref]. It is evident that sonoporation has the potential to play a large role in the future of regenerative medicine but will benefit from a greater foundation of pre-clinical research.
Author Contributions: Writing-original draft preparation, Z.K.; writing-review and editing, Z.K., Z.G. and G.P.; supervision-D.G. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by the U.S. Department of Defense, grant number W81XWH-18-1-0593, and by the NIH/NIBIB, grant number R01EB026094.
[fig] Figure 1: Image depicting the induction of a pore in the cell membrane by cavitation of microbubbles, allowing exogenous nucleic acids to passively diffuse into the cytoplasm. Created with Bio-Render (BioRender.com, accessed on 20 April 2022). [/fig]
[table] Table 1: Advantages and disadvantages of non-viral gene delivery methods. [/table]
[table] Table 3: The use of sonoporation for skeletal tissue regeneration. [/table]
[table] Table 4: The use of sonoporation for cardiac tissue regeneration. [/table]
[table] Table 5: The use of sonoporation for ischemia in peripheral tissue. [/table]
[table] Table 6: The use of sonoporation as a therapy for diabetes. [/table]
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Automated Glycan Assembly in a Variable-Bed Flow Reactor Provides Insights into Oligosaccharide–Resin Interactions
## General information
All reagents and solvents were acquired from commercial sources, unless stated otherwise. Thioglycoside monosaccharide building block was purchased from GlycoUniverse. Conversion to mannosyl phosphate donor 1 were prepared according to literature procedures.Pentaacetatyled mannosyl phosphate donor matched that of previously reported.HPLC grade solvents were used. Resin was prepared according to literature.ESI-HRMS were performed with a Xevo G2-XS Q-Tof (Waters). Anhydrous solvents were prepared by drying over activated molecular sieves. NMR spectra were obtained using Ascend 400 (Bruker) and Agilent 400 MHz NMR Magnet (Agilent Technologies) spectrometers at 400 MHz ( 1 H) and 100 MHz ( 13 C). CDCl 3 was used as solvent and chemical shifts (δ) referenced to internal standards (CDCl 3 : 7.26 ppm 1 H, 77.16 ppm 13 C) unless stated otherwise. Assignments were supported by COSY and HSQC experiments. Only diagnostic signals are listed. For monitoring reactions by mass spectrometry, an Agilent 1100 Series LC/MSD mass spectrometer was used. HPLCs were performed on Agilent 1200 Series systems. A YMC-Diol-300-NP column (150 mm x 4.60 mm I.D.) was used for analytical NP-HPLC, with a flow rate of 1.00 mL/min and hexanes:EtOAc as eluent. A YMC-Pack Diol-300-NP column (150 mm x 20.0 mm I.D.) was used for preparative NP-HPLC, with a flow rate of 15.0 mL/min and hexanes:EtOAc as eluent. Unless stated otherwise, the gradient program detailed hereafter was used: 1. Isocratic 20% EtOAc in hexanes (5 min). 2. Linear gradient 20 to 55% EtOAc in hexanes (35 min). 3. Linear gradient to 100% EtOAc (10 min). UV-vis (312 nm) and VBFR data were processed using Origin software.
## S4
# Additional figures
## General synthetic procedures
## Oligosaccharide synthesis
Resin Preparation: Desired resin (0.1 g, 0.33 mmol g -1 ) was loaded into the Omnifit column and the pistons were put into place. The VBFR was set to a resin bed height of 1.5 cm and resin was allowed to swell by flowing CHCl 3 from pumps A and B at a flow rate of 0.3 mL min -1 . After 10 min with the pumps still flowing, the cooling coils were then cooled to -10 °C for an additional 5 min.
Once at temperature (-10 °C) the VBFR was set to achieve a differential pressure of 8 bar to pack the resin. Pumps A and B continued to pass CHCl 3 (0.3 mL/min) over until synthesis commenced.
Building Block Preparation: Mannosyl donor 1 was separated into individual 20 mL vials with each containing enough material for one cycle (270 mg). Each vial was then co-evaporated with toluene three times and placed under high vacuum overnight. Building block stock solutions (2 mL, 0.15 M) in anhydrous CHCl 3 were prepared and transferred into individual 10 mL microwave reactor vessels with a crimped septum under argon. Stock activator solutions were made by addition of 2 mL of anhydrous CHCl 3 into individual 10 mL microwave reactor vessels with a crimped septum under argon. The appropriate amount of TMSOTf was then added (60 µL, 0.15 M) to each activator vessel (one for every glycosylation cycle). Solutions were placed in proper autosampler position.
Modules for Automated Synthesis:
## S8
## Oligosaccharide resin cleavage and purification
After completion of AGA, the resin was transferred into a 10 mL fritted syringe and washed with CH 2 Cl 2 and dried in vacuo. The Vapourtec E-Series UV-150 Photoreactor Flow Chemistry System was employed. The medium pressure metal halide lamp was filtered using the commercial longpass filter (No. 3, red). The resin, suspended in CH 2 Cl 2 , was loaded into a 20 mL plastic syringe. The suspension was pumped using a syringe pump (PHD2000, Harvard Apparatus) at 0.7 mL min -1 through a 10 mL reactor, constructed of 1/8inch o.d. FEP tubing. The temperature of the photoreactor was maintained at 20 °C and the lamp power was adjusted to 80%. The exiting flow was filtered and the filtrate was collected into a collection flask. The crude oligosaccharide was dried in vacuo, then dissolved in 50:50 hexanes:ethyl acetate and analyzed by analytical HPLC (Agilent 1200). Crude mixture was purified with preparative NP-HPLC.
## Preliminary optimization study
TMSOTf (1 equiv)
[formula] CHCl 3 , -10°C O O FmocO BnO BnO OBz P O O BnO BnO OBz NHCbz HO O BuO OBu 1% DBU CHCl 3 , 20°C [/formula]
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A pressure-resistant peripherally inserted central catheter is as useful as a central venous catheter for rapid fluid infusion: an in vitro study
Background: Although peripherally inserted central catheters (PICCs) have been widely used, they have not been frequently used in anesthesia practice. The central venous pressure measured via PICCs are reportedly as accurate as that measured via central venous catheters (CVCs), but the findings concerning rapid infusion are unclear. This study examined whether or not pressure-resistant PICCs could be used for rapid fluid infusion.Methods:The in-line pressure was measured in similar-sized double-lumen catheters-4-Fr PICC (55, 45 and 35 cm) and 17-G CVC (20 and 13 cm)-at flow rates of saline decided using a roller pump system. We also examined the flow rate at an in-line pressure of 300 mmHg, which is the critical pressure at which hemolysis is considered to occur during blood transfusion.Results:The pressure-resistant PICCs obtained a high flow rate similar to that of CVCs, but the in-line pressures increased in proportion to the flow rate and catheter length. Flow rates at an intra-circuit pressure of 300 mmHg were not significantly different between the 45-cm PICC and 20-cm CVC.Conclusion: Pressure-resistant PICCs can be used for rapid fluid infusion.
# Introduction
Central venous catheters (CVCs) are used in perioperative management to monitor central venous pressure (CVP), administer cardiovascular agents and drugs irritating to veins, perform rapid fluid infusion and transfuse blood. However, because CVC insertion is associated with severe mechanical complications, the indications of CVCs are limited [bib_ref] Indications for peripheral, midline and central catheters: summary of the MAGIC recommendations, Moureau [/bib_ref]. In contrast, the insertion of a peripherally inserted central catheter (PICC) is safer and easier than that of a CVC according to a retrospective study [bib_ref] Safety and feasibility of ultrasound-guided placement of peripherally inserted central catheter performed..., Kim [/bib_ref]. Although PICCs are widely used for parenteral nutrition, chemotherapy, and long-term administration of antimicrobial agents, they have not been frequently used in perioperative management or acute care, as the accuracy of the CVP measured via a PICC and the reliability of PICCs as a route for rapid fluid infusion are unclear.
In hemodynamic monitoring, the CVP is measured via a CVC in 19% of non-cardiac surgeries lasting longer than 90 min and is useful for hepatobiliary surgeries [bib_ref] Safety and feasibility of ultrasound-guided placement of peripherally inserted central catheter performed..., Kim [/bib_ref] [bib_ref] Methods to decrease blood loss during liver resection: a network metaanalysis, Moggia [/bib_ref] , kidney transplantation [bib_ref] Fluid Management During Kidney Transplantation: A Consensus Statement of the Committee on..., Wagener [/bib_ref]. In both in vitro and clinical studies, the CVP measured via a PICC has been reported to be as accurate as that measured via a CVC Open Access *Correspondence: [email protected] [bib_ref] An in vitro study comparing a peripherally inserted central catheter to a..., Latham [/bib_ref] [bib_ref] Central venous pressure monitoring via peripherally or centrally inserted central catheters: a..., Sanfilippo [/bib_ref]. Therefore, the use of a PICC to monitor the CVP in anesthetic management and acute care is suggested to be possible.
As an intravenous route for rapid infusion and blood pressure, CVCs are also useful in clinical practice [bib_ref] Peripherally inserted central catheter placement in patients with coagulation disorders: A retrospective..., Potet [/bib_ref]. However, if PICCs are used as a rapid infusion route, inline pressure may rise too much due to longer line route. In recent years, pressure-resistant PICCs suitable for injection of contrast media have become available. Therefore, in the present study, we examined whether or not pressure-resistant PICCs could be used for rapid fluid infusion as quickly as CVCs in vitro.
# Methods
The catheters examined were as follows: 55-cm 4-Fr (outer diameter 1.33 mm) dual-lumen PICC (Power PICC ™ ; Bard Access Systems, Inc. Salt Lake City, UT, USA), and a 20-and 13-cm 17-G (outer diameter 1.35 mm) dual-lumen CVC (SMAC ™ plus; Cardinal Health, Dublin, OH, USA) [fig_ref] Table 1: Details of the PICC and CVC PICC Peripherally inserted central catheter, CVC... [/fig_ref]. After measurement, the 55-cm PICC was cut to 45 and 35 cm, in sequence.
Regarding the connection of the PICC and CVC to the infusion sets, the infusion circuit used to measure in-line pressure consisted of a blood transfusion set (TB-PU300L; TERUMO, Tokyo, Japan), the roller pump segment of a hemodialysis circuit (NS-1010-20; NIPRO, Osaka, Japan), and a three-way stopcock (L1-360FL; TOP, Tokyo, Japan). The blood transfusion set was connected to a 500-mL bag of normal saline solution, and the 3-way stopcock was connected to the main lumen of a dual-lumen catheter (a PICC or CVC). The circuit was attached to a roller pump system for hemodialysis (MP-301; NIPRO). A pressure gauge (PG-208-103GP-S; NIDEC COPAL ELECTRON-ICS, INC., Tokyo, Japan) was placed at the three-way stopcock to measure the in-line pressure. The second lumen of the catheter was connected to a bag of normal saline via another blood transfusion set and perfused with 20 mL/h normal saline using an infusion pump (TE-261; TERUMO), which simulated the use of a second lumen as a route for the administration of drugs such as catecholamines. The catheter tip was then placed 10 cm under the surface of normal saline solution in a cup open in the atmosphere [fig_ref] Figure 1: A schematic illustration of infusion circuit for measuring in-line pressure [/fig_ref].
The flow rate of normal saline solution infused via the main lumen was regulated by the roller pump and increased and decreased stepwise by 10 mL/min between 0 and 150 mL/min. After the equilibration period, the maximum in-line pressure was recorded at each flow rate. The in-line pressure at each flow rate was measured four times in each catheter.
The data were expressed as the average ± standard error of the mean of values obtained from three catheters. The data were analyzed using a t-test or one-way
# Results
In-line pressures increased with the flow rate of normal saline at 0 to 150 mL/min in both the PICC and CVC [fig_ref] Figure 2: Relationships between the flow rates and in-line pressure [/fig_ref]. The maximum pressures in the 45-cm PICC and 20-cm CVC were 2084 ± 62 and 2523 ± 13 mmHg, respectively. In-line pressures increased in proportion to the length of catheter in both PICC and CVC [fig_ref] Figure 2: Relationships between the flow rates and in-line pressure [/fig_ref]. On comparing the 45-cm PICC and 20-cm CVC, the inline pressures were higher in the 45-cm PICC at a lowflow range of 10 to 30 mL/min but lower in the 45-cm PICC at a high-flow range of 90 to 150 mL/min [fig_ref] Figure 2: Relationships between the flow rates and in-line pressure [/fig_ref]. Data comparing the 55-cm PICC and 20-cm CVC are shown in Additional file 2.
The flow rates when the in-line pressure was ≤ 300 mmHg decreased in proportion to the catheter length in both the PICC and CVC (data not shown). The flow rate when the in-line pressure was 300 mmHg was not significantly different between a 45-cm PICC and 20-cm CVC [fig_ref] Figure 3: A comparison of the flow rates at an in-line pressure of 300... [/fig_ref] but was lower in the 55-cm PICC than in the 20-cm CVC (Additional file 3).
The in-line pressure was not affected by repeated measurement (Additional file 1).
# Discussion
In the present study, flow rates of 0-150 mL/min were obtained in the pressure-resistant PICC and CVC. However, the in-line pressures were positively correlated with the catheter length. The flow rate at in-line pressures < 300 mmHg, when hemolysis is expected to occur, did not markedly differ between the 45-cm pressureresistant PICC and 20-cm CVC.
Regarding the relationship between the flow rate and in-line pressure, longer catheters have a higher in-line pressure than shorter ones. In our previous report concerning the relationship between the flow rate and inline pressure using a peripheral venous catheter, there was a positive correlation between the catheter length and in-line pressure with an 18-G peripheral venous catheter, which has same diameter as the lumen of the PICC used in the present study. The in-line pressure was approximately 300 mmHg at a flow rate of 150 mL/min (9000 mL/h) [bib_ref] In-line pressure within a HOTLINE(R) Fluid Warmer, under various flow conditions, Higashi [/bib_ref]. The length of the peripheral venous catheter was 4.8 cm in the previous study, while the lengths of the CVC and PICC with the same diameter in the present study were 20 and 13 cm and 55, 45 and 35 cm. In each catheter, the in-line pressure increased to 1745-2523 mmHg as the catheter length was increased. However, in the present study, we confirmed that repeated infusions at an in-line pressure of ≥ 2000 mmHg did not affect the flow rate or in-line pressure using a pressure-resistant PICC or CVC. These results suggest that there is no issue with performing repeated high-pressure infusion.
Compared with rapid infusion from a peripheral venous catheter, the flow rate was limited when rapid intravenous infusion was performed with a PICC. However, a pressure-resistant PICC can withstand rapid intravenous infusion of 150 mL/min (9000 mL/h), which is clinically sufficient. The maximum pressure of a clinical pressurized rapid infusion device is limited to 300 mmHg, and it has been reported that in-line pressure is increased to about 600 mmHg during manual rapid infusion using a piston syringe [bib_ref] The properties of an improvised piston pump for the rapid delivery of..., Smart [/bib_ref]. In the present study, a pressure-resistant PICC was able to withstand a higher pressure than exerted with these methods, suggesting that a pressure-resistant PICC can be safely used for rapid infusion.
On comparing a 45-cm PICC with a 20-cm CVC, which are usually used clinically, the PICC, which was longer than the CVC, was expected to have a higher in-line pressure with the same flow rate provided the catheter diameter was the same. However, in actuality, the in-line pressure of the PICC was higher than that of the CVC at a low flow rate, whereas the in-line pressure of the PICC at a high flow rate was significantly lower than that of the CVC. This is considered to be due to the lumen partition wall of the double-lumen tube used in this study. In CVCs, the luminal septum does not move, whereas in pressure-resistant PICCs, the shape of the catheter lumen changes according to the in-line pressure, which expands the effective inner diameter of the catheter.
Regarding red blood cell products, hemolysis reportedly occurs under pressures exceeding 300 mmHg [bib_ref] Expediting red blood cell transfusions by syringing causes significant hemolysis, Villiers [/bib_ref]. Thus, rapid transfusion using a pressurized rapid transfusion device was performed at a maximum internal pressure of 300 mmHg. In the present study, the rate of saline administration that resulted in an in-line pressure of 300 mmHg did not differ between the 45-cm PICC and 20-cm CVC and was comparable to 30 mL/ min (1800 mL/h). Therefore, the pressure-resistant PICC can be used as a transfusion route as well as the CVC in case of rapid transfusion due to massive bleeding. If the flow rate is higher than this, transfusion should be performed through a larger venous route, such as a peripheral venous catheter or sheath.
Several limitations associated with the present study warrant mention. First, the data in the present study were obtained from an in vitro study. Thus, the in-line pressure may be influenced by the intravascular position and resistance of vasculature and may actually be much higher than the result shown here. Second, because the in-line pressure was not measured using red blood cells, the usefulness of a pressure-resistant PICC in blood transfusion has not been confirmed.
# Conclusion
Pressure-resistant PICCs can be used for rapid fluid infusion as fast as CVCs.
## Abbreviations
CVCs: Central venous catheters; CVP: Central venous pressure; PICCs: Peripherally inserted central catheter.
## Supplementary information
The online version contains supplementary material available at https:// doi. org/ 10. 1186/ s12871-022-01738-x.
Additional file 1. Changes in the in-line pressures after repeated measurements.
Additional file 2. In-line pressures at the indicated flow rates in the 55-cm PICC (gray triangles) were compared with those in CVCs (filled circles). Data are shown as the average ± SEM, and significant differences are represented by * (p < 0.05).
[fig] Figure 1: A schematic illustration of infusion circuit for measuring in-line pressure. The circuit to infuse normal saline consisted of a blood transfusion set, a roller pump system and a three-way stopcock connected to the main lumen of a catheter. A pressure gauge was connected to the stopcock. The second lumen of the catheter was perfused with normal saline using an infusion pump [/fig]
[fig] Figure 2: Relationships between the flow rates and in-line pressure. (a) Triangles and (b) circles show in-line pressures at indicated flow rates in 55-, 45-and 35-cm peripherally inserted central catheters (PICCs) and 20-and 13-cm central venous catheters (CVCs). (c) In-line pressures at the indicated flow rates in the 45-cm PICC (gray triangles) were compared with those in CVCs (filled circles). Data are shown as the average ± SEM, and significant differences are represented by * (p < 0.05) [/fig]
[fig] Figure 3: A comparison of the flow rates at an in-line pressure of 300 mmHg. The flow rates at an in-line pressure of 300 mmHg were compared between a 20-cm central venous catheter (CVC) and 45-cm peripherally inserted central catheter (PICC). The flow rates were not significantly different between these catheters (p < 0.05). Data are shown as the average ± SEM. n.s., not significantly different [/fig]
[table] Table 1: Details of the PICC and CVC PICC Peripherally inserted central catheter, CVC Central venous catheter, OD Outer catheter diameter, ID Inner catheter diameter analysis of variance with Tukey's test. A p-value < 0.05 was considered statistically significant. [/table]
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Natural history of inflammatory bowel disease: a comparison between the East and the West
# Introduction
Inflammatory bowel disease (IBD) was previously considered a rare disease in Asia. However, over the past few decades, there has been a rapid increase in its incidence and prevalence, posing a substantial healthcare burden in this area. [bib_ref] A 30-year trend analysis in the epidemiology of inflammatory bowel disease in..., Park [/bib_ref] [bib_ref] The epidemiology of inflammatory bowel disease: east meets west, Mak [/bib_ref] [bib_ref] Epidemiological trend in inflammatory bowel disease in Taiwan from 2001 to 2015:..., Yen [/bib_ref] [bib_ref] Marshall JK. Prevalence and patient awareness of inflammatory bowel disease in Kazakhstan:..., Kaibullayeva [/bib_ref] Characteristics of IBD in Asian populations are known to be different from those in Western populations in many aspects. [bib_ref] An update on the epidemiology of inflammatory bowel disease in Asia, Thia [/bib_ref] Therefore, to adequately cope with this chronic and progressive disease, it is important to fully understand the unique clinical course of IBD in Asian populations.
The clinical course of IBD may be influenced by many factors, including disease phenotypes and genotypes, delays in www.irjournal.org reported that 27%-45% of patients with CD received anti-tuberculosis therapy before they were finally diagnosed with CD because of the difficulty in differentiating between CD and intestinal tuberculosis, resulting in diagnostic delay and increased risk of stenotic complications. [bib_ref] Risk factors for diagnostic delay in Crohn' s disease and their impact..., Banerjee [/bib_ref] [bib_ref] Current status and clinical characteristics of inflammatory bowel disease in Korea, Yang [/bib_ref] In addition, the high prevalence of infectious diseases, including hepatitis B virus infection, [bib_ref] Prevention and management of viral hepatitis in inflammatory bowel disease: a clinical..., Park [/bib_ref] [bib_ref] Clinical courses of chronic hepatitis B virus infection and inflammatory bowel disease..., Park [/bib_ref] tuberculosis, [bib_ref] Asian Organization for Crohn' s and Colitis and Asian Pacific Association of..., Park [/bib_ref] [bib_ref] Risk of incident Mycobacterium tuberculosis infection in patients with inflammatory bowel disease:..., Hong [/bib_ref] and cytomegalovirus colitis, [bib_ref] Review of cytomegalovirus seroprevalence and demographic characteristics associated with infection, Cannon [/bib_ref] [bib_ref] Severe disease activity and cytomegalovirus colitis are predictive of a nonresponse to..., Park [/bib_ref] in Asian countries may adversely affect the treatment of IBD. Third, there may be racial differences in the medication response and the adverse events associated with medications. Thiopurine-induced acute severe leukopenia is more common in Asians than in Caucasians because of the higher prevalence of NUDT15 variants, limiting the use of thiopurines in Asians. [bib_ref] A common missense variant in NUDT15 confers susceptibility to thiopurine-induced leukopenia, Yang [/bib_ref] [bib_ref] NUDT15 gene variants and thiopurine-induced leukopenia in patients with inflammatory bowel disease, Matsuoka [/bib_ref] [bib_ref] Best practices on immunomodulators and biologic agents for ulcerative colitis and Crohn'..., Ooi [/bib_ref] In contrast, previous studies have suggested that the response to biologic agents, such as infliximab, [bib_ref] Long-term prognosis of Japanese patients with biologic-naïve Crohn' s disease treated with..., Moroi [/bib_ref] [bib_ref] Long-term outcomes of infliximab treatment in 582 Korean patients with Crohn' s..., Park [/bib_ref] [bib_ref] Long-term outcomes of infliximab treatment and predictors of response in 195 patients..., Seo [/bib_ref] adalimumab, [bib_ref] Long-term outcomes of adalimumab treatment in 254 patients with Crohn' s disease:..., Seo [/bib_ref] [bib_ref] Long-term outcomes of adalimumab therapy in Korean patients with ulcerative colitis: a..., Oh [/bib_ref] [bib_ref] Long-term safety and effectiveness of adalimumab in Japanese patients with Crohn' s..., Hisamatsu [/bib_ref] vedolizumab, [bib_ref] Efficacy and safety of vedolizumab in ulcerative colitis in patients from Asian..., Ooi [/bib_ref] [bib_ref] Efficacy and safety of vedolizumab in Crohn' s disease in patients from..., Banerjee [/bib_ref] [bib_ref] Population pharmacokinetics of vedolizumab in Asian and non-Asian patients with ulcerative colitis..., Okamoto [/bib_ref] and ustekinumab, [bib_ref] Efficacy and safety of ustekinumab in East Asian patients with moderately to..., Hisamatsu [/bib_ref] does not differ between Asian and Western patients with IBD. Fourth, although the introduction of biologic agents has dramatically changed the paradigm of IBD treatment and they may be equally efficacious in Asian and Western patients, their use in Asian countries is limited because of the high cost and the generally unpermitted top-down therapy. Accordingly, a simple comparison of disease outcomes, such as surgical rates, without considering the confounding effects of the aforementioned fac-
# Methods
## Outcomes of interest
To describe the natural history of IBD in Asian patients, we evaluated several IBD-related outcomes including phenotypic progression, hospitalization, major surgery, risk of colorectal cancers, and mortality. Moreover, when data is available, the temporal trends of IBD-related clinical outcomes in Asian patients with IBD were assessed. The outcomes of these Asian studies were compared with those of Western populationbased cohort studies [fig_ref] Table 1: Outcomes of Patients with Ulcerative Colitis in Asian Population-Based Studies in Contrast... [/fig_ref]. If necessary, results from hospital-based studies were mentioned.
## Literature search
We conducted a systematic literature search in MEDLINE using the PubMed database to identify mainly population-based studies and, in case of lacking population-based studies, hospital-based studies from Asian countries which reported the natural history of IBD patients. We searched for the following terms: ("inflammatory bowel disease" OR "ulcerative colitis" OR "Crohn' s disease") AND ("population-based" OR "nationwide") AND ("natural history" OR "natural course" OR "longterm outcome" OR "long-term follow-up" OR "temporal trend" OR "temporal change" OR "phenotype" OR "progression" OR "extension" OR "surgery" OR "resection" OR "colectomy" OR "perianal" OR "fistula" OR "hospitalization" OR "cancer" OR "malignancy" OR "mortality"
# Results
## Phenotypic progression
## 1) uc
The extent of disease upon UC diagnosis is known to be different between Western and Asian patients. In a systematic review of 17 population-based cohorts, [bib_ref] Natural history of adult ulcerative colitis in population-based cohorts: a systematic review, Fumery [/bib_ref] the most common disease presentation at diagnosis was left-sided colitis (median, 40.1%; interquartile range [IQR], 32.6%-44.6%), followed by extensive colitis (30.5%; IQR, 29.8%-32.6%) and proctitis (29.4%; IQR, 25.3%-34.7%). In contrast, in the SK-IBD cohort, the most common disease presentation at diagnosis was proctitis (54.3%), followed by extensive colitis (23.2%) and left-sided colitis (22.5%). [bib_ref] A 30-year trend analysis in the epidemiology of inflammatory bowel disease in..., Park [/bib_ref] Similarly, in the ACCESS cohort with patients from 8 Asian countries, disease presentation at diagnosis was proctitis (36.5%), extensive colitis (32.8%), and left-sided colitis (30.7%) in decreasing order of proportion. [bib_ref] Early course of inflammatory bowel disease in a population-based inception cohort study..., Ng [/bib_ref] During the disease course, patients with UC experience proximal progression of the disease. Proximal disease extension appeared to carry a more severe disease course and was associated with an increased need for immunosuppressive treatment and an increased risk of hospitalization and colectomy. [bib_ref] Ulcerative colitis as a progressive disease: the forgotten evidence, Torres [/bib_ref] In a population-based cohort study from Veszprem Province, Hungary, the cumulative risks of proximal disease extension at 1, 3, 5 years after the diagnosis of ulcerative proctitis or left-sided colitis were 2.9%, 9.4%, and 12.7%, respectively. [bib_ref] Incidence, disease phenotype at diagnosis, and early disease course in inflammatory bowel..., Lakatos [/bib_ref] In a European population-based inception cohort (the Epi-IBD cohort), 90 (20.7%) of 435 patients with proctitis or Proximal disease extension in ulcerative colitis and disease behavior progression in Crohn's disease. SIR, standardized incidence ratio; SMR, standardized mortality ratio; CI, confidence interval; RR, relative risk; HR, hazard ratio. www.irjournal.org left-sided colitis at diagnosis experienced proximal disease extension during the first 5 years of follow-up. [bib_ref] Natural disease course of ulcerative colitis during the first five years of..., Burisch [/bib_ref] In a Danish population-based inception cohort, 72 (32.7%) of 220 patients with proctitis or left-sided colitis at diagnosis experienced proximal disease extension during the median follow-up of 7.5 years. [bib_ref] Proximal disease extension in patients with limited ulcerative colitis: a Danish population-based..., Burisch [/bib_ref] Additionally, in a population-based inception cohort of South-Eastern Norway (the IBSEN cohort), 100 (34.7%) of 288 patients with proctitis or left-sided colitis at diagnosis experienced proximal disease extension during the first 10 years of followup. [bib_ref] Clinical course during the first 10 years of ulcerative colitis: results from..., Solberg [/bib_ref] Meanwhile, in a meta-analysis of 30 studies including both population-based and hospital-based studies and both Western and Asian studies, the cumulative risk of proximal disease extension was 17.8% (95% confidence interval [CI], 12.3-25.1) at 5 years and 31.0% (95% CI, 23.5-39.7) at 10 years. [bib_ref] Systematic review with meta-analysis: proximal disease extension in limited ulcerative colitis, Roda [/bib_ref] In a population-based cohort study from Orebro, Sweden, between 1963 and 2010, there was no difference in the risk of proximal disease extension among the 3 temporal cohorts. [bib_ref] Changes in medical management and colectomy rates: a population-based cohort study on..., Eriksson [/bib_ref] In the SK-IBD cohort, the cumulative risks of proximal disease extension at 5, 10, 20, and 30 years after the diagnosis of ulcerative proctitis or left-sided colitis were 20.5%, 30.2%, 46.7%, and 54.0%, respectively. [bib_ref] Long-term prognosis of ulcerative colitis and its temporal changes between 1986 and..., Cha [/bib_ref] The risks of proximal disease extension were significantly higher in patients with proctitis than in those with left-sided colitis at diagnosis and did not differ among the 3 temporal cohorts. [bib_ref] Long-term prognosis of ulcerative colitis and its temporal changes between 1986 and..., Cha [/bib_ref] In the ACCESS cohort, the cumulative risk of proximal disease extension in Asian patients was 15.3% at 2-3 years after diagnosis. [bib_ref] Early course of inflammatory bowel disease in a population-based inception cohort study..., Ng [/bib_ref] Taken together, the risk of proximal disease extension in Asian patients is comparable with that in Western patients. However, it remains unknown whether any treatment can reduce the risk of proximal disease extension. The results of the SK-IBD cohort study showing no change in the risk of proximal disease extension between 1986 and 2015 raise questions about the role of any treatment in reducing the risk of proximal disease extension. Further studies using standardized approaches are required to investigate proximal disease extension.
## 2) cd
Although the most common disease behavior at CD diagnosis is inflammatory behavior, Western population-based cohort studies have demonstrated that 15%-35% of patients with CD already had stricturing or penetrating behavior at the time of CD diagnosis. [bib_ref] Incidence, disease phenotype at diagnosis, and early disease course in inflammatory bowel..., Lakatos [/bib_ref] [bib_ref] Natural disease course of Crohn' s disease during the first 5 years..., Burisch [/bib_ref] [bib_ref] Changes in disease behaviour and location in patients with Crohn' s disease..., Lo [/bib_ref] [bib_ref] Surgery in a population-based cohort of Crohn' s disease from Olmsted County, Peyrin-Biroulet [/bib_ref] [bib_ref] Epidemiology and long-term outcome of inflammatory bowel disease diagnosed at elderly age-an..., Jeuring [/bib_ref] [bib_ref] The Faroese IBD Study: incidence of inflammatory bowel diseases across 54 years..., Hammer [/bib_ref] [bib_ref] Natural history of Crohn' s disease in a population-based cohort from Cardiff..., Ramadas [/bib_ref] [bib_ref] Temporal trends in non-stricturing and non-penetrating behaviour at diagnosis of Crohn' s..., Zhulina [/bib_ref] In the SK-IBD cohort, disease behavior at CD diagnosis was inflammatory in 81.1%, stricturing in 8.1%, and penetrating in 10.8%. [bib_ref] A 30-year trend analysis in the epidemiology of inflammatory bowel disease in..., Park [/bib_ref] In a population-based cohort study from Singapore, disease behavior at CD diagnosis was inflammatory in 78.1%, stricturing in 14.0%, and penetrating in 7.9%. [bib_ref] Risk of major abdominal surgery in an Asian population-based Crohn' s disease..., Pandey [/bib_ref] In the ACCESS cohort, disease behavior at CD diagnosis was inflammatory in 68.1%, stricturing in 18.8%, and penetrating in 13.0%. [bib_ref] Early course of inflammatory bowel disease in a population-based inception cohort study..., Ng [/bib_ref] Perianal fistulas are common manifestations of CD and are known to be a predictor of poor prognosis. Perianal fistulas are considered more common in Asian patients with CD than in Western patients with CD. [bib_ref] Ananthakrishnan AN. Ethnicity influences phenotype and outcomes in inflammatory bowel disease: a..., Shi [/bib_ref] In Western population-based cohort studies, 8.1%-16.7% of patients with CD developed a perianal fistula before or at CD diagnosis. [bib_ref] The natural history of fistulizing Crohn' s disease in Olmsted County, Minnesota, Schwartz [/bib_ref] [bib_ref] Risk factors associated with progression to intestinal complications of Crohn' s disease..., Thia [/bib_ref] [bib_ref] Update on the natural course of fistulizing perianal Crohn' s disease in..., Park [/bib_ref] [bib_ref] Occurrence and outcome after primary treatment of anal fistulae in Crohn' s..., Hellers [/bib_ref] [bib_ref] Incidence and clinical course of Crohn' s disease during the first year:..., Sjöberg [/bib_ref] [bib_ref] A 20-year temporal change analysis in incidence, presenting phenotype and mortality, in..., Van Den Heuvel [/bib_ref] In contrast, 43.3% and 33.3% of patients with CD developed a perianal fistula before or at CD diagnosis in the SK-IBD cohort 1 and a Korean nationwide population-based cohort study using the National Health Insurance claims data, [bib_ref] Incidence and outcomes of perianal disease in an Asian population with Crohn'..., Song [/bib_ref] respectively. In addition, in the Hong Kong IBD Registry, 20.9% of patients with CD had perianal CD before or at CD diagnosis. [bib_ref] Significant medical and surgical morbidity in perianal Crohn' s disease: results from..., Mak [/bib_ref] In population-based cohort studies from Europe, 14%-22% of patients with CD who had an inflammatory behavior at diagnosis experienced disease behavior progression during the first 5 years after diagnosis. [bib_ref] Natural disease course of Crohn' s disease during the first 5 years..., Burisch [/bib_ref] [bib_ref] Changes in disease behaviour and location in patients with Crohn' s disease..., Lo [/bib_ref] [bib_ref] Improvements in the long-term outcome of Crohn' s disease over the past..., Jeuring [/bib_ref] In a population-based cohort study from Olmsted County, the cumulative risks of disease behavior progression at 1, 5, 10, 20, and 30 years after the diagnosis of CD with inflammatory behavior were 4.1%, 18.5%, 24.7%, 39.5%, and 43.9%, respectively. [bib_ref] Risk factors associated with progression to intestinal complications of Crohn' s disease..., Thia [/bib_ref] Regarding the temporal trends of disease behavior progression, the Dutch IBD South Limburg cohort failed to show any changes between 1991 and 2011. [bib_ref] Improvements in the long-term outcome of Crohn' s disease over the past..., Jeuring [/bib_ref] However, in a population-based inception cohort from Western Hungary, the 5-year cumulative risk of disease behavior progression significantly decreased from 35.5% in the 1977-1989 cohort to 15.2% in the 1999-2008 cohort. [bib_ref] Has there been a change in the natural history of Crohn' s..., Lakatos [/bib_ref] Regarding perianal fistulas, in Western population-based cohort studies, 13.9%-28.1% of patients with CD developed perianal fistulas during the median follow-up of 8.4-16.2 years after CD diagnosis. [bib_ref] The natural history of fistulizing Crohn' s disease in Olmsted County, Minnesota, Schwartz [/bib_ref] [bib_ref] Risk factors associated with progression to intestinal complications of Crohn' s disease..., Thia [/bib_ref] [bib_ref] Update on the natural course of fistulizing perianal Crohn' s disease in..., Park [/bib_ref] [bib_ref] Occurrence and outcome after primary treatment of anal fistulae in Crohn' s..., Hellers [/bib_ref] [bib_ref] Time trends in the epidemiology and outcome of perianal fistulizing Crohn' s..., Göttgens [/bib_ref] [bib_ref] Risk factors for surgery and postoperative recurrence in Crohn' s disease, Bernell [/bib_ref] In the Dutch IBD South Limburg cohort, the cumulative risks of developing a perianal fistula at 1, 5, and 10 years after CD diagnosis were 8.3%, 11.6%, and 15.8%, respectively. [bib_ref] Time trends in the epidemiology and outcome of perianal fistulizing Crohn' s..., Göttgens [/bib_ref] In a population-based cohort study from Olmsted County, the cumulative risks of developing a perianal fistula at 1, 5, 10, 20, and 30-40 years after CD diagnosis were 11%, 15%, 18%, 23%, and 24%, respectively. [bib_ref] Update on the natural course of fistulizing perianal Crohn' s disease in..., Park [/bib_ref] In the SK-IBD cohort, the cumulative risks of disease behavior progression at 1, 5, 10, and 20 years after the diagnosis of CD with inflammatory behavior were 9.2%, 19.3%, 32.5%, and 54.5%, respectively. [bib_ref] Changes in the long-term prognosis of Crohn' s disease between 1986 and..., Ye [/bib_ref] Compared with the 1986-2003 cohort, the 2004-2015 cohort had significantly lower cumulative risks of progression: 6.9%, 16.4%, and 23.2% at 1, 5, and 10 years, respectively. [bib_ref] Changes in the long-term prognosis of Crohn' s disease between 1986 and..., Ye [/bib_ref] In the ACCESS cohort, the cumulative risk of behavioral progression in Asian patients was 20.4% at 2-3 years www.irjournal.org after diagnosis. [bib_ref] Early course of inflammatory bowel disease in a population-based inception cohort study..., Ng [/bib_ref] Taken together, there appears to be no difference in the risk of disease behavior progression between Asian and Western patients with CD. As for perianal fistulas, in a population-based cohort study using the National Health Insurance claims data, 39.2% of patients with CD developed a perianal fistula during the median follow-up of 4.2 years after CD diagnosis. [bib_ref] Incidence and outcomes of perianal disease in an Asian population with Crohn'..., Song [/bib_ref] The cumulative risks of developing a perianal fistula at 1 and 5 years after CD diagnosis were 35.2% and 40.0%, respectively. In addition, in the Hong Kong IBD Registry, 28.8% of patients with CD had perianal disease during the median follow-up of 8.8 years after CD diagnosis. [bib_ref] Significant medical and surgical morbidity in perianal Crohn' s disease: results from..., Mak [/bib_ref]
## Hospitalization
## 1) uc
In a recent review of European population-based cohort studies, the 1-and 5-year hospitalization rates in patients with UC were 9%-33% and 18%-54%, respectively. [bib_ref] The burden of inflammatory bowel disease in Europe in 2020, Zhao [/bib_ref] Hospitalization rates varied substantially among different European countries. [bib_ref] The burden of inflammatory bowel disease in Europe in 2020, Zhao [/bib_ref] In the Olmsted County cohort, the cumulative risk of hospitalization at 5, 10, 20, and 30 years after UC diagnosis was 29.4%, 38.7%, 49.2%, and 52.3%, respectively. [bib_ref] Cumulative incidence and risk factors for hospitalization and surgery in a populationbased..., Samuel [/bib_ref] In addition, the rate of hospitalization in the Olmsted County cohort decreased from 134/1,000 patient-years in 1970-1979 to 88/1,000 patient-years in 2000-2004. [bib_ref] Cumulative incidence and risk factors for hospitalization and surgery in a populationbased..., Samuel [/bib_ref] In the SK-IBD cohort, the cumulative risks of hospitalization at 1, 5, and 10 years after UC diagnosis were 10.6%, 15.1%, and 18.4%, respectively. [bib_ref] Long-term prognosis of ulcerative colitis and its temporal changes between 1986 and..., Cha [/bib_ref] The cumulative risk of hospitalization was significantly lower in the recent cohort than in the older cohort, with the 5-year cumulative risk of hospitalization being 25.3% in the 1986-1999 cohort and 12.5% in the 2010-2015 cohort. This temporal trend of decreasing hospitalization rate was also observed in a hospital-based cohort study from Thailand, where the cumulative risks of hospitalization at 1 and 5 years after diagnosis were 18% and 30%, respectively, in the 2000-2009 cohort and 5% and 21%, respectively, in the 2010-2018 cohort. [bib_ref] Temporal trend in the natural history of ulcerative colitis in a country..., Aniwan [/bib_ref] Furthermore, considering the absolute risks of hospitalization, it appears that Asian patients with UC have lower risks compared with Western patients. However, this does not necessarily mean that Asian patients experience a less severe disease form because the hospitalization rate is influenced by many factors other than disease severity, such as accessibility to healthcare services.
## 2) cd
In a recent review of European population-based cohort studies, the 1-and 5-year hospitalization rates in patients with CD were 23%-49% and 44%-54%, respectively. [bib_ref] The burden of inflammatory bowel disease in Europe in 2020, Zhao [/bib_ref] In the Olmsted County cohort, 120 (57%) of 211 patients with CD had at least 1 CD-related hospitalization during the total observation period of 2,247 patient-years. [bib_ref] Hospitalization rates for Crohn' s disease patients in Olmsted county, Minnesota, in..., Ingle [/bib_ref] In the Dutch IBD South Limburg cohort, the risk of hospitalization in patients with CD significantly decreased over time, with the 5-year cumulative risk of hospitalization being 65.9% in the 1991-1998 cohort and 44.2% in the 2006-2011 cohort. [bib_ref] Improvements in the long-term outcome of Crohn' s disease over the past..., Jeuring [/bib_ref] In a population-based study from Ontario, Canada, CD-related hospitalizations decreased by 32% from 154/1,000 patients in 2003 to 104/1,000 patients in 2014. [bib_ref] Declining hospitalisation and surgical intervention rates in patients with Crohn' s disease:..., Rahman [/bib_ref] In the SK-IBD cohort, the cumulative risks of hospitalization at 1, 5, 10, and 20 years after CD diagnosis were 31.2%, 40.7%, 51.9%, and 67.9%, respectively. [bib_ref] Long-term prognosis of ulcerative colitis and its temporal changes between 1986 and..., Cha [/bib_ref] The cumulative risk of hospitalization was significantly lower in the recent cohort than in the older cohort, with the 5-year cumulative risk of hospitalization being 52.2% in the 1986-2003 cohort and 36.6% in the 2004-2015 cohort. The cumulative risks of hospitalization in the SK-IBD cohort were similar to or slightly lower than those in Western population-based cohort studies. [bib_ref] The burden of inflammatory bowel disease in Europe in 2020, Zhao [/bib_ref] [bib_ref] Improvements in the long-term outcome of Crohn' s disease over the past..., Jeuring [/bib_ref] [bib_ref] Hospitalization rates for Crohn' s disease patients in Olmsted county, Minnesota, in..., Ingle [/bib_ref] [bib_ref] Declining hospitalisation and surgical intervention rates in patients with Crohn' s disease:..., Rahman [/bib_ref]
## Major surgery
## 1) uc
In a meta-analysis of 26 population-based cohort studies from Europe (n = 17), North America (n = 5), Oceania (n = 2), Asia (n = 1), and Africa (n = 1), the cumulative risks of colectomy at 1, 5, and 10 years after UC diagnosis were 4.0% (95% CI, 3.3-5.0), 8.8% (95% CI, 7.7-10.0), and 13.3% (95% CI, 11.3-15.5), respectively. [bib_ref] Contemporary risk of surgery in www.irjournal.org patients with ulcerative colitis and Crohn'..., Tsai [/bib_ref] Among patients diagnosed in the 21st century, the cumulative risks of colectomy at 1, 5, and 10 years after UC diagnosis were 2.8% (95% CI, 2.0-3.9), 7.0% (95% CI, 5.7-8.6), and 9.6% (95% CI, 6.3-14.2), respectively. There was no difference in the cumulative risk of colectomy between Europe and North America. [bib_ref] Contemporary risk of surgery in www.irjournal.org patients with ulcerative colitis and Crohn'..., Tsai [/bib_ref] Compared with Western studies, population-based cohort studies from Asia have reported very low colectomy rates. [bib_ref] Long-term prognosis of ulcerative colitis and its temporal changes between 1986 and..., Cha [/bib_ref] [bib_ref] Early course of inflammatory bowel disease in a population-based inception cohort study..., Ng [/bib_ref] [bib_ref] Epidemiology of inflammatory bowel disease from 1981 to 2014: results from a..., Ng [/bib_ref] The cumulative risk of colectomy at 1 year after UC diagnosis was only 1.1% in the ACCESS cohort, [bib_ref] Early course of inflammatory bowel disease in a population-based inception cohort study..., Ng [/bib_ref] and that at 1 and 5 years after UC diagnosis was 1.8% and 2.1%, respectively, in the Hong Kong IBD Registry. [bib_ref] Epidemiology of inflammatory bowel disease from 1981 to 2014: results from a..., Ng [/bib_ref] The risk of colectomy in the SK-IBD cohort was even lower than that reported in these 2 studies. In the SK-IBD cohort, the cumulative risks of colectomy at 1, 5, 10, 20, and 30 years after UC diagnosis were 1.0%, 1.9%, 2.2%, 5.1%, and 6.4%, respectively. [bib_ref] Long-term prognosis of ulcerative colitis and its temporal changes between 1986 and..., Cha [/bib_ref] Because the cumulative risks of colectomy significantly decreased over the study period of 1986-2015, they were even lower among patients diagnosed www.irjournal.org in the 21st century: 0.4% at 1 year, 1.4% at 5 years, and 1.6% at 10 years after diagnosis. Moreover, in the 2010-2015 cohort, no patients underwent colectomy during the first year after diagnosis, and only 0.8% of patients underwent colectomy during the first 5 years after diagnosis. [bib_ref] Long-term prognosis of ulcerative colitis and its temporal changes between 1986 and..., Cha [/bib_ref]
## 2) cd
In a meta-analysis of 22 population-based cohort studies from Europe (n = 16), North America (n = 2), Oceania (n = 2), Asia (n = 1), and Africa (n = 1), the cumulative risks of intestinal resection at 1, 5, and 10 years after CD diagnosis were 18.7% (95% CI, 15.0-23.0), 28.0% (95% CI, 24.0-32.4), and 39.5% (95% CI, 33.3-46.2), respectively. [bib_ref] Contemporary risk of surgery in www.irjournal.org patients with ulcerative colitis and Crohn'..., Tsai [/bib_ref] Among patients diagnosed in the 21st century, the cumulative risks of intestinal resection at 1, 5, and 10 years after CD diagnosis were 12.3% (95% CI, 10.8-14.0), 18.0% (95% CI, 15.4-21.0), and 26.2% (95% CI, 23.4-29.4), respectively. There was no difference in the cumulative risk of intestinal resection between Europe and North America. [bib_ref] Contemporary risk of surgery in www.irjournal.org patients with ulcerative colitis and Crohn'..., Tsai [/bib_ref] Proctectomy is required in some patients with a refractory complex perianal fistula. In a population-based cohort study from Olmsted County, the cumulative risk of proctectomy was 20% at 10 years and 22% at 20-40 years after the diagnosis of a perianal fistula. [bib_ref] Update on the natural course of fistulizing perianal Crohn' s disease in..., Park [/bib_ref] In the SK-IBD cohort, the cumulative risks of intestinal resection at 1, 5, 10, 20, and 25 years after CD diagnosis were 12.7%, 16.5%, 23.8%, 45.1%, and 51.2%, respectively, with a trend of decreasing risk over the study period. [bib_ref] Long-term prognosis of ulcerative colitis and its temporal changes between 1986 and..., Cha [/bib_ref] In the 2004-2015 cohort, the cumulative risks of intestinal resection at 1, 5, and 10 years after CD diagnosis were 11.4%, 15.1%, and 18.7%, respectively. In a population-based study from Singapore, the cumulative risks of intestinal resection at 5, 10, 20, and 30 years after CD diagnosis were 21.2%, 28.8%, 38.3%, and 50.6%, respectively. [bib_ref] Risk of major abdominal surgery in an Asian population-based Crohn' s disease..., Pandey [/bib_ref] In the Hong Kong IBD Registry, the cumulative risks of intestinal resection at 1 and 5 years after CD diagnosis were 20.3% and 25.7%, respectively. [bib_ref] Epidemiology of inflammatory bowel disease from 1981 to 2014: results from a..., Ng [/bib_ref] The cumulative risk of intestinal resection at 1 year after CD diagnosis was the lowest at 8.0% in the ACCESS cohort. [bib_ref] Early course of inflammatory bowel disease in a population-based inception cohort study..., Ng [/bib_ref] Taken together, the cumulative risks of intestinal resection at 1, 5, and 10 years after CD diagnosis in Asian population-based cohort studies were 8%-20%, 17%-26%, and 24%-29%, respectively. These rates are comparable to or slightly lower than those of the aforementioned metaanalysis. In addition, the cumulative risks of intestinal resection in the 2004-2015 cohort of the SK-IBD cohort were comparable to or slightly lower than those in the contemporary cohorts of patients diagnosed with CD after 2000 in the metaanalysis. As for proctectomy due to a perianal fistula, in the Hong Kong IBD Registry, the cumulative risks of defunctioning surgery and/or proctectomy at 1, 3, and 10 years after the diagnosis of a perianal fistula were 4.1%, 5.4%, and 6.8%, respectively. [bib_ref] Significant medical and surgical morbidity in perianal Crohn' s disease: results from..., Mak [/bib_ref] In a hospital-based cohort from Korea, the cumulative risks of proctectomy at 10, 20, and 30 years after the diagnosis of a perianal fistula were 2.9%, 12.2%, and 16.2%, respectively. [bib_ref] Incidence and outcomes of perianal disease in an Asian population with Crohn'..., Song [/bib_ref] Therefore, the risk of proctectomy in patients with CD who developed perianal fistulas is lower in Asian patients than in Western patients. In this Korean study, the cumulative risk of proctectomy was significantly lower in the biologic era than in the pre-biologic era. [bib_ref] Incidence and outcomes of perianal disease in an Asian population with Crohn'..., Song [/bib_ref]
## Risk of colorectal cancer
## 1) uc
In a meta-analysis of 8 Western population-based cohort studies from Europe (n = 6) and North America (n = 2), the cumulative risks of colorectal cancer were < 1.0% at 10 years, 0.4%-2.0% at 15 years, and 1.1%-5.3% at 20 years after UC diagnosis, and the pooled standardized incidence ratio (SIR) for colorectal cancer was 2.4 (95% CI, 2.1-2.7). [bib_ref] Risk of colorectal cancer in patients with ulcerative colitis: a meta-analysis of..., Jess [/bib_ref] In population-based cohort studies from Copenhagen and Hungary, the 30-year cumulative risks of colorectal cancer were 2.1% and 7.5%, respectively. [bib_ref] Longterm risk of cancer in ulcerative colitis: a population-based cohort study from..., Winther [/bib_ref] [bib_ref] Risk factors for ulcerative colitis-associated colorectal cancer in a Hungarian cohort of..., Lakatos [/bib_ref] In a study involving the Uppsala and Stockholm cohorts of IBD, there was a trend toward a decrease in the risk of colorectal cancer from 1960 to 2004, but it did not reach statistical significance. [bib_ref] Decreasing time-trends of colorectal cancer in a large cohort of patients with..., Söderlund [/bib_ref] However, in a study involving a nationwide cohort of Danish patients with IBD, the relative risk of colorectal cancer in patients with UC decreased significantly from 1.34 (95% CI, 1.13-1.58) in 1979-1988 to 0.57 (95% CI, 0.41-0.80) in 1999-2008. [bib_ref] Decreasing risk of colorectal cancer in patients with inflammatory bowel disease over..., Jess [/bib_ref] Recently, 3 population-based studies from Asian countries have been published on the risk of colorectal cancer in patients with IBD. In the Hong Kong IBD Registry, during the median follow-up of 10 years, 13 of 1,603 patients with UC developed colorectal cancer, with an SIR of 0.95 (95% CI, 0.55-1.64). [bib_ref] Cancer risk in 2621 Chinese patients with inflammatory bowel disease: a population-based..., So [/bib_ref] Similarly, in a study from Taiwan, during the mean follow-up of 7.2 years, 18 of 2,663 patients with UC developed colorectal cancer, with an SIR of 1.39 (95% CI, 0.8-2.2). [bib_ref] Higher risk for hematological malignancies in inflammatory bowel disease: a nationwide population-based..., Wang [/bib_ref] No observed increase in the risk of colorectal cancer in these 2 studies was possibly due to the short follow-up period. In contrast, in a study from Korea utilizing the National Health Insurance claims data, during the median follow-up of 2.20 years (range, 0.00-3.99 years), 31 of 9,785 patients with UC developed colorectal cancer, with SIRs of 2.14 (95% CI, 1.31-3.30) for male patients and 2.95 (95% CI, 1.47-5.29) for female patients. [bib_ref] Cancer risk in the early stages of inflammatory bowel disease in Korean..., Jung [/bib_ref] Although this Korean study demonstrated an increased risk of colorectal cancer in patients www.irjournal.org with UC, considering the very short follow-up period, the results of this Korean study should be interpreted with caution. According to previous Danish population-based studies, there was a remarkably high risk of colorectal cancer in the first year after IBD diagnosis, probably due to coincidental detection of recent-onset or prevalent IBD in patients diagnosed with colorectal cancer and misclassification of IBD versus cancer. [bib_ref] Decreasing risk of colorectal cancer in patients with inflammatory bowel disease over..., Jess [/bib_ref] [bib_ref] Risk of cancer in patients with inflammatory bowel diseases: a nationwide population-based..., Kappelman [/bib_ref] Therefore, more population-based studies with a longer follow-up period are needed to verify these results. In hospitalbased cohort studies, the 30-year cumulative risk of colorectal cancer in Asian patients with UC ranged from 5.2% to 14.4%, [bib_ref] The risk of colorectal cancer in inflammatory bowel disease: a hospital-based cohort..., Lee [/bib_ref] [bib_ref] Results of a 36-year surveillance program for ulcerative colitis-associated neoplasia in the..., Kishikawa [/bib_ref] [bib_ref] Risk of ulcerative colitis-associated colorectal cancer in China: a multi-center retrospective study, Gong [/bib_ref] [bib_ref] Long-term follow-up of ulcerative colitis in Taiwan, Wei [/bib_ref] which was similar to that in previous Western studies. In a hospital-based cohort study from Korea, the SIR for colorectal cancer in patients with UC was 1.70 (95% CI, 1.00-2.66). [bib_ref] The risk of colorectal cancer in inflammatory bowel disease: a hospital-based cohort..., Lee [/bib_ref]
## 2) cd
In a meta-analysis of 6 population-based cohort studies from Europe (n = 3), North America (n = 2), and Israel (n = 1), the pooled SIR for colorectal cancer was 1.9 (95% CI, 1.4-2.5). [bib_ref] Increased risk of intestinal cancer in Crohn' s disease: a metaanalysis of..., Jess [/bib_ref] In a study involving a nationwide cohort of Danish patients with IBD, the relative risk of colorectal cancer in patients with CD was lower than previously known (relative risk, 0.85; 95% CI, 0.67-1.07) and, in contrast to UC, did not decrease significantly from 1979 to 2008. [bib_ref] Decreasing risk of colorectal cancer in patients with inflammatory bowel disease over..., Jess [/bib_ref] In the 3 Asian population-based cohort studies, the SIRs for colorectal cancer in patients with CD were 1.64 (95% CI, 0.74-3.65) in Hong Kong, 37 0.96 (95% CI, 0.1-3.5) in Taiwan, [bib_ref] Higher risk for hematological malignancies in inflammatory bowel disease: a nationwide population-based..., Wang [/bib_ref] [bib_ref] Cancer risk in the early stages of inflammatory bowel disease in Korean..., Jung [/bib_ref] These results have problems similar to that in UC. In a hospital-based cohort study from Korea, the SIR for colorectal cancer in patients with CD was 6.0 (95% CI, 3.10-10.48). [bib_ref] The risk of colorectal cancer in inflammatory bowel disease: a hospital-based cohort..., Lee [/bib_ref] Of note, in all 6 patients who developed colorectal cancer in the Hong Kong IBD Registry, the cancer was located in the anorectum. [bib_ref] Cancer risk in 2621 Chinese patients with inflammatory bowel disease: a population-based..., So [/bib_ref] Similarly, in hospital-based cohort studies from Korea 79 and Japan, 84,85 the colorectal cancer was located in the rectum in 89%-100% of patients with CD and colorectal cancer, in contrast with only 40% in Western studies. [bib_ref] Declining risk of colorectal cancer in inflammatory bowel disease: an updated meta-analysis..., Lutgens [/bib_ref] The high proportion of rectal cancer in Asian patients with CD and colorectal cancer may be partly explained by the high prevalence of perianal fistulas in this population. 1,79,87
## Mortality
## 1) uc
Most Western population-based cohort studies published after 2000 reported no increase in mortality in patients with UC compared with in the general population, [bib_ref] Ulcerative colitis: no rise in mortality in a European-wide population based cohort..., Höie [/bib_ref] [bib_ref] Mortality and causes of death in ulcerative colitis: results from 20 years..., Hovde [/bib_ref] [bib_ref] Mortality in inflammatory bowel disease in the Netherlands 1991-2002: results of a..., Romberg-Camps [/bib_ref] [bib_ref] Mortality in ulcerative colitis and Crohn' s disease: a population-based study in..., Manninen [/bib_ref] [bib_ref] Total and cancer mortality in a cohort of ulcerative colitis and Crohn'..., Caini [/bib_ref] barring some studies that reported a slight increase [bib_ref] Trends in overall and cause-specific mortality among patients with inflammatory bowel disease..., Jess [/bib_ref] [bib_ref] Mortality trends in Crohn' s disease and ulcerative colitis: a population-based study..., Bitton [/bib_ref] or decrease. [bib_ref] Overall and cause-specific mortality of inflammatory bowel disease in Olmsted County, Aniwan [/bib_ref] In a meta-analysis of 10 Western population-based cohort studies from Europe (n = 8), North America (n = 1), and Oceania (n = 1), the pooled standardized mortality ratio (SMR) was 1.1 (95% CI, 0.9-1.2). [bib_ref] Overall and cause-specific mortality in ulcerative colitis: meta-analysis of population-based inception cohort..., Jess [/bib_ref] In a study involving a Danish national cohort, the mortality in patients with UC was significantly higher than that in matched controls (hazard ratio, 1.25; 95% CI, 1.22-1.28), and it decreased gradually from 1982 to 2010. [bib_ref] Trends in overall and cause-specific mortality among patients with inflammatory bowel disease..., Jess [/bib_ref] In the SK-IBD cohort, the cumulative survival rates at 1, 5, 10, 20, and 30 years after UC diagnosis were 99.7%, 98.7%, 96.6%, 92.2%, and 91.2%, respectively, and the SMR was 0.725 (95% CI, 0.508-1.004). [bib_ref] Long-term prognosis of ulcerative colitis and its temporal changes between 1986 and..., Cha [/bib_ref] In the Hong Kong IBD Registry, the SMR was 1.5 (95% CI, 0.9-2.3). [bib_ref] Epidemiology of inflammatory bowel disease from 1981 to 2014: results from a..., Ng [/bib_ref] In contrast to these 2 Asian studies and the Western meta-analysis, a population-based study from Taiwan using the National Health Insurance database showed increased mortality in patients with UC (SMR, 1.44; 95% CI, 1.26-1.65). [bib_ref] Trends and risk factors of mortality analysis in patients with inflammatory bowel..., Lin [/bib_ref] The SMR in the Taiwanese patients with UC decreased from 1.88 (95% CI, 1.33-2.60) in 2001-2005 to 1.36 (95% CI, 1.11-1.65) in 2011-2015. However, the mortality rate remained higher in patients with UC than in the general population even in the most recent cohort. The reason for this high mortality rate is unclear. This may be explained by inadequate treatment due to the low awareness of this rare disease, misclassification bias inherent in code-based administrative database studies, or differences in prognosis among different populations.
## 2) cd
In contrast to UC, a meta-analysis of 9 Western populationbased cohort studies from Europe (n = 8) and North America (n = 1) demonstrated increased mortality from CD with a pooled SMR of 1.39 (95% CI, 1. . [bib_ref] Overall and cause-specific mortality in Crohn' s disease: a meta-analysis of population-based..., Duricova [/bib_ref] In addition, in a Danish national cohort, there was no improvement in the hazard ratio for mortality in patients with CD from 1982 to 2010. [bib_ref] Trends in overall and cause-specific mortality among patients with inflammatory bowel disease..., Jess [/bib_ref] In the SK-IBD cohort, the cumulative survival rates at 1, 5, 10, 20, and 30 years after CD diagnosis were 100%, 99.0%, 98.6%, 98.6%, and 84.5%, respectively, and the SMR was 1.36 (95% CI, 0.59-2.68). [bib_ref] Long-term prognosis of ulcerative colitis and its temporal changes between 1986 and..., Cha [/bib_ref] In the Hong Kong IBD Registry, the SMR was 2.0 (95% CI, 0.9-4.0). [bib_ref] Epidemiology of inflammatory bowel disease from 1981 to 2014: results from a..., Ng [/bib_ref] The SMRs in these 2 Asian studies seemed comparable to those in Western studies, although the increased mortality from CD in these 2 studies did not reach statistical significance probably due to a type II error. In contrast to these 2 Asian studies, a population-based study from Taiwan showed a remarkably increased mortality from CD (SMR, 3.72; 95% CI, 3.02-4.55). [bib_ref] Trends and risk factors of mortality analysis in patients with inflammatory bowel..., Lin [/bib_ref] The SMR in the Taiwanese patients with CD decreased from 5.46 (95% CI, 3.38-8.43) in 2001-2005 to 2.80 www.irjournal.org (95% CI, 2.01-3.80) in 2011-2015. However, the SMR in the most recent cohort remained higher than that in the Western meta-analysis. Possible reasons for this high mortality were discussed in the section on UC mortality.
# Summary and conclusion
The results of this review suggest that the clinical course of Asian patients with IBD, especially UC, is better than that of Western patients, as indicated by the lower rates of major surgery and hospitalization. In addition, as in Western patients, the clinical course of Asian patients with IBD is improving, as evidenced by decreasing rates of disease behavior progression (in CD), hospitalization, and major surgery.
However, this study had a few limitations. First, this review contains data only from East Asia and Southeast Asia, because there are no population-based studies on the clinical course of IBD in the Middle East and South Asia. Therefore, it is unclear whether the disease outcomes of this review can be generalized to all Asian populations, considering that there is genotypic and phenotypic heterogeneity among IBD patients in different Asian populations. [bib_ref] An update on the epidemiology of inflammatory bowel disease in Asia, Thia [/bib_ref] [bib_ref] Association analyses identify 38 susceptibility loci for inflammatory bowel disease and highlight..., Liu [/bib_ref] [bib_ref] Changing epidemiological trends of inflammatory bowel disease in Asia, Ng [/bib_ref] Second, although Asian countries have very heterogeneous medical environments, the different medical standards of each country were not considered in this review. Third, we could not reach conclusions in the case of some outcome parameters such as colorectal cancer risk and mortality rate, because these parameters need longerterm studies.
Therefore, to make a definite conclusion on the prognosis of IBD in Asian patients, more studies from different Asian countries with long-term follow-up are needed. Knowledge of the clinical course of Asian patients with IBD may help in selecting optimal treatment strategies and developing healthcare policies.
[table] Table 1: Outcomes of Patients with Ulcerative Colitis in Asian Population-Based Studies in Contrast to a Systematic Review and Meta-Analysis from Western Population-Based Studies IQR, interquartile range; CI, confidence interval; SK-IBD, Songpa-Kangdong Inflammatory Bowel Disease; ACCESS, Asia-Pacific Crohn's and Colitis Epidemiology Study. www.irjournal.org tors, may lead to false conclusions on the prognosis of Asian patients with IBD.In this review, we aimed to evaluate how the clinical course of IBD in Asian patients differs from that in Western patients and investigate whether there are temporal trends toward improvement in the clinical course of Asian patients with IBD. [/table]
[table] Table 2: Outcomes of Patients with Crohn's Disease in Asian Population-Based Studies in Contrast to a Systematic Review and Meta-Analysis from Western Population-Based Studies [/table]
[table] Table 3: Comparison of Outcomes of Patients with Inflammatory Bowel Disease between the East and the West along with the Temporal Trends [/table]
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Levels of serum estradiol and lifestyle factors related with bone mineral density in premenopausal Mexican women: a cross-sectional analysis
Background: Many factors, such as heredity, ethnicity, nutrition and other lifestyle factors, have been related to bone mineral density in postmenopausal women. Additionally, bone mass has been significantly associated with decreased estrogen levels. However, fewstudies have been conducted on premenopausal women. The present study was designed to estimate the relationship between low bone mineral density and levels of serum estradiol and lifestyle factors in premenopausal Mexican women.Methods:A cross-sectional study was conducted in 270 women between 40 and 48 years of age who participate in the Health Workers Cohort Study. Information on socio-demographic and lifestyle factors were obtained through a self-administered questionnaire. Body mass index and serum estradiol were measured with standard procedures; bone mineral density was assessed using dual-energy X-ray absorptiometry. Multiple linear and logistic regression models were computed to evaluate the relationship between low bone mineral density and levels of serum estradiol and lifestyle factors.Results:In linear regression analysis levels of estradiol, body mass index, physical activity, and vitamin D intake were positively related to bone mineral density. Age, cigarette smoking and caffeine were inversely associated with BMD. Finally, the odds of low bone mineral density increase significantly when the premenopausal women had low levels of serum estradiol (OR = 4.93, 95 % CI: 2.14, 11.37).Conclusion: These data support that low serum estradiol, advancing age, lower physical activity, lower vitamin D intake, cigarette smoking, and higher amount of caffeine intake are linked to low bone mineral density in premenopausal Mexican women.
# Background
Osteoporosis is a skeletal condition represented by low bone mineral density and the depreciation of the microarchitecture of the bone tissue [bib_ref] Clinician's guide to prevention and treatment of osteoposis, Cosman [/bib_ref]. While this disorder is common among the elderly, it is also a problem among younger people. For example, in premenopausal women, about 15 % have bone mineral density levels below −1 standard deviation, and about 0.6 % are more than −2.5 standard deviations below young-adult mean bone mineral density [bib_ref] Low bone mineral density in premenopausal women, Lewiecki [/bib_ref]. Globally, osteoporosis is one of the most important public health problems, predisposing people to fractures [bib_ref] Adverse outcomes of osteoporotic fractures in the general population, Melton 3rd [/bib_ref] [bib_ref] Prevention and treatment of non-postmenopausal osteoporosis, Hansen [/bib_ref] [bib_ref] Epidemiology and outcomes of osteoporotic fractures, Cummings [/bib_ref] [bib_ref] Diagnosis of osteoporosis and assessment of fracture risk, Kanis [/bib_ref] , which are associated with increased morbidity and mortality [bib_ref] Adverse outcomes of osteoporotic fractures in the general population, Melton 3rd [/bib_ref] [bib_ref] Prevention and treatment of non-postmenopausal osteoporosis, Hansen [/bib_ref] , reduced quality of life [bib_ref] Epidemiology and outcomes of osteoporotic fractures, Cummings [/bib_ref] and pose high health-care costs [bib_ref] Diagnosis of osteoporosis and assessment of fracture risk, Kanis [/bib_ref].
Bone mineral density (BMD) in women has been related to many factors such as age at menarche, number of pregnancies, breastfeeding, and contraceptive use; as well as physical activity, tobacco use and intakes of calcium, caffeine, dietary fiber, dietary vitamin D and alcohol [bib_ref] A meta-analysis of the effects of cigarette smoking on bone mineral density, Ward [/bib_ref] [bib_ref] To drink or not to drink: How are alcohol, caffeine and past..., Ilich [/bib_ref] [bib_ref] Influence of number of pregnancies on bone mineral density in posmenopausal women..., Gur [/bib_ref] [bib_ref] Cigarete smoking, alcohol consumption, and risk of Hip fracture in women, Baron [/bib_ref] [bib_ref] Associations of calcium intake and physical activity with bone density and size..., Uusi [/bib_ref].
Perimenopause is an intermediate phase in a woman's life when physiological changes occur that begin the transition to menopause, such as irregular menstrual periods [bib_ref] A longitudinal study of the effect of genistein on bone in two..., Reinwald [/bib_ref] [bib_ref] Associations between Low Levels of Serum Estradiol, Bone Density, and Fractures among..., Ettinger [/bib_ref]. In Mexican women, it has been observed that after the age of 40 years bone mass decreases gradually [bib_ref] Bone speed of sound throughout lifetime assessed with quantitative ultrasound in a..., Rivas-Ruiz [/bib_ref] , which could be associated with lowered estrogen production that happens several years prior to the start of the menopause. On the other hand, estrogens play an important role in bone growth and homeostasis [bib_ref] Editorial: local biosynthesis of sex steroids in bone, Compston [/bib_ref]. Furthermore, in women, levels of sex steroids are a significant component responsible for bone turnover and bone density [bib_ref] Estrone sulfate is a major source of local estrogen formation in human..., Muir [/bib_ref] , especially when estrogen production reduces and levels fall, accelerated bone loss ensues.
Therefore, our study was designed to evaluate the relationship between bone mineral density and levels of serum estradiol and lifestyle factors in premenopausal Mexican women.
# Methods
## Study population
The present study included a sample of adult females (40-48 years old) participating in the Health Workers Cohort Study (HWCS). In short, the HWCS include healthy members from health and academic institutions located in two cities of central Mexico: Instituto Mexicano del Seguro Social (IMSS) and the Instituto Nacional de Salud Pública (INSP) and Universidad Autónoma del Estado de México (UAEMex). In general, the main objective of the HWCS is to evaluate the associations between different lifestyle factors and health outcomes in the Mexican population. The specifics about methods and participants' characteristics have been described previously [bib_ref] Tabaquismo en profesionales de la salud del Instituto Mexicano del Seguro Social, Salmeron-Castro [/bib_ref] [bib_ref] Predictors of bone mineral density in female workers in Morelos State, López-Caudana [/bib_ref] [bib_ref] Reference values for areal bone mineral density among a healthy Mexican population, Tamayo [/bib_ref].
From March 2004 to April 2006, a total of 9,467 subjects were requested to participate in the HWCS, of these, 8,307 adults were registered in the cohort study. Of those, the female population of the HWCS consists of 6,240 women ranging from 18 to 90 years. Due to the nature of the study, only women working at the UAEMex were included, individuals with the following characteristics were excluded from the final analysis: females who work at IMSS, or at INSP (n = 4392); women under 40 years and over 48 years (n = 1439); subjects reporting current or past occurrence of any medical conditions recognized to influence bone metabolism (n = 26); and women who had been taking any medication that affects bone density, for example, glucocorticoids (n = 84). We also excluded subjects who did not have complete information (n = 29). In total, 270 females were included in the final analysis. All of them continued with regular menstrual cycles.
All women participating in the study gave written informed consent to the procedures, which were reviewed and accepted by the ethics committee of the Medical Science Research Center (No. 1233008X0236). Finally, the present study was conducted following the recommendations of the Declaration of Helsinki.
## Measures
Females included in the present study gave information on socio-demographic characteristics, reproductive factors (such as: number of pregnancies, breastfeeding, and contraceptive use) and lifestyle information on dietary intake, use of multivitamin supplements, physical activity, tobacco use and family history of osteoporosis.
## Physical activity measurement
The International Physical Activity Questionnaire (IPAQ) [bib_ref] Compendium of Physical Activities: an update of activity codes and MET intensities, Ainsworth [/bib_ref] , which has been previously adjusted for the Mexican population, was used to assess habitual physical activity. Subjects answered questions concerning the period of time they assigned to physical activity throughout leisure time, at work and doing housework. Physical activity was measured in metabolic equivalents (METS) and was estimated according to the time and intensity performed in different activities like walking, dancing, swimming, and doing other exercises for 1 week, in the year previous to the study. We have also evaluated participants' sedentary activities, defined as time devoted to activity with low METS. Sedentary activities included reading, writing, working at the computer, watching TV, going to the movies, activity planning and playing table games.
## Dietary intake measurement
A food frequency questionnaire (FFQ) previously validated in Mexican adult population [bib_ref] Validity and reproducibility of a food frequency questionnaire to assess dietary intake..., Hernández-Avila [/bib_ref] was employed to evaluate dietary intake. The FFQ included information on the average frequency of intake of 116 food items during the previous year. Dietary vitamin D (UI/d) as well as dietary calcium (mg/d) consumption were estimated using the FFQ above mentioned. Multivitamin supplement use (including calcium) was determined accordant to names and daily doses reported by participants. Calcium and vitamin D intake were determined by the sum of dietary calcium consumption plus vitamin D and calcium from supplement consumption. Finally, both, the consumption of dietary vitamin D and calcium were estimated by multiplying the frequency of consumption of each food by the vitamin D and calcium content of the specified portion size. Additionally, Alcohol and caffeine consumption were assessed as drinks/day, from which g/day and mg/day, respectively, were calculated. By means of a food contents database we computed the specific nutrient intakes.
Using the standard deviation method suggested by Rosner [bib_ref] Percentage points for a generalized ESD many-outlier procedure, Rosner [/bib_ref] , the outlier values in energy intake (values below 600 kcal/day and above 7000 kcal/day) were eliminated from the final analysis. All nutrient intakes were adjusted by the residual method.
Tobacco use and familial history of osteoporosis Tobacco use was assessed by means of a self-reported questionnaire, constructed on previous and current cigarette smoking. Tobacco use was categorized as "yes" for current smoking and past smoking, or "no" for never smoking. Additionally, a female was defined as a smoker, if she informed consuming at least 100 cigarettes in her lifetime [bib_ref] Tabaquismo en profesionales de la salud del Instituto Mexicano del Seguro Social, Salmeron-Castro [/bib_ref] [bib_ref] Predictors of bone mineral density in female workers in Morelos State, López-Caudana [/bib_ref].
If a woman responded yes to the question "Has anyone in your family ever had osteoporosis?" She was classified as a positive to familial history of osteoporosis [bib_ref] Predictors of bone mineral density in female workers in Morelos State, López-Caudana [/bib_ref].
## Reproductive factors measurement
The reproductive factors like: age at menarche, duration of menstrual cycles, use of contraceptive, number of gestations, and duration of breastfeed were assessed. Age at menarche was defined as the age of the first menstrual bleeding. Duration of menstrual cycles was described as the time in days from the first day of menstruation to the initiation of the following period. In our study population all women continued with their menstrual cycle. The use of oral contraceptive was expressed as the accrued time since women took oral contraceptives. We obtained information about the number of gestations (including only full-term pregnancies), which were dichotomized as < 2, and ≥ 2 gestations, to the final analysis. Lastly, the time of breastfeed was assessed by the calculation of total months that a woman gave breastfeeding to her different children, for the present analysis, two groups were established: < 6 months and ≥ 6 months [bib_ref] Reproductive and lifestyle factors associated with early menopause in Mexican women, Ortega-Ceballos [/bib_ref].
## Anthropometric measurements
Body weight (kg) was evaluated with an electronic TANITA scale (model BC-533, Tokyo, Japan), formerly calibrated, with females dressing minimal clothes. Body height (m) was assessed by means of a stadiometer. Using a measuring tape, waist circumference was calculated at the high point of the iliac crest at the end of normal expiration; to the nearest 0.1 cm. Body mass index was calculated as a ratio of weight (kg) to height in meters squared (BMI, kg/m 2 ). For the present study, BMI was utilized to categorize women as follows: normal BMI (≥18.5 to <25.0 kg/m 2 ); overweight (≥25.0 to <30.0 kg/m 2 ) and obese (≥30.0 kg/m 2 ).
## Levels of serum estradiol
A blood sample was drawn between 7 and 9 am into evacuated tubes after subjects fasted overnight (at least 12 h). Levels of serum estradiol (pg/mL) were assessed through competitive immunoassay (Immulite estradiol, Diagnostic Products Corp., Los Angeles, CA). Immulite is a computerized, random-access immunoassay analyzer with a solid-phase and a competitive chemiluminescent enzyme. The solid stage (bead) is covered with rabbit anti-estradiol polyclonal antibody. The women's serum test and alkaline phosphataseconjugated are concurrently insert into the test unit. During 60-min incubation at 37°C with intermittent shaking, estradiol in the patient sample competes with the enzyme-labeled estradiol for a partial quantity of antibody binding sites on the bead. Chemiluminescent substrate, a phosphate ester of adamantyl dioxetane, is inserted and the test unit is incubated for 10 min. The substrate is hydrolyzed by alkaline phosphatase to an unstable anion. The disintegration of the anion yields a sustained release of light. The bound complex, corresponding to the photon output, is inversely proportional to the relative amount of estradiol in the sample. A single establishment involves 25 μL of serum, and the stated working range of the test is 20 to 2000 pg/mL. The antibodies employed in the Immulite estradiol assay are secondary to the estradiol molecule at the specificity-enhancing sixth position. The improved specificity of this 17 β-estradiol antiserum makes quantification of estradiol amounts more precise. Reference ranges were used to define women with low estradiol levels (<60 pg/mL) and women with normal concentrations of estradiol (≥60 pg/mL) [bib_ref] Minimal levels of serum estradiol prevent postmenopausal bone loss, Reginster [/bib_ref].
## Bone mineral density measurements
Bone mineral density determinations were carrying out at the non-dominant proximal femur (FNBMD), the lumbar spine (L1-L4) (LSBMD), and on the whole body (TBMD) with a dual-energy X-ray absorptiometry (DXA, Lunar DPX NT instrument). The guidelines established by the International Society of Clinical Densitometry were strictly followed [bib_ref] Official Positions of the International Society for Clinical Densitometry, Leib [/bib_ref]. Densitometry technicians performed all BMD measurements using standardized procedures. Customary calibration of the densitometer was accomplish every day by the phantom delivered by the manufacturer; specialists guaranteed that the day-today coefficient of variation (CV) was lower than 1.5 %. DXA measurements are presented as grams of hydroxyapatite per square centimeter. For the present study, the sample of females was divided into two categories according to the standards suggested by the World Health Organization (WHO): normal if T-score > − 1.0 and low bone mineral density; defined as follow: osteoporosis if Tscore ≤ −2.5; and osteopenia if −2.5 < T-score ≤ −1.0 [bib_ref] Identification and fracture outcomes of undiagnosed Low bone mineral density in postmenopausal..., Siris [/bib_ref].
# Statistical analysis
We conducted a descriptive analysis of the principal characteristics of interest of the study population. We contrasted predefined risk factors of BMD and levels of BMD between low and normal levels of serum estradiol in premenopausal women. We used the t-student to compare continuous variables with a normal distribution. While, in continuous variables with non-normal distributions, the Mann-Whitney U test was used. In addition, to compare categorical variables between groups (low vs. normal) chi square test was computed.
To assess the effects of levels of estradiol and other risk factors on bone mineral density, we computed multiple linear regression analysis.
We computed adjusted odds ratio (OR) and 95 % confidence interval (95 % CI) by means of multiple logistic regression models to determine the size of the relationship between low levels of serum estradiol and other risk factors and bone mineral density.
P values lower than 0.05 were thoughtful as statistically significant. All analyses were conducted with STATA software, version 13.0 (Stata Corp LP, College Station, Tex, USA). [fig_ref] Table 1: Characteristics of the study population by levels of serum estradiol [/fig_ref]. Participants' mean age was 43.8 years (SD = 2.6), and their mean BMD was 1.18 (SD = 0.08) g/cm 2 .
# Results
## A description of subjects' characteristics is shown in
Premenopausal women with low levels of serum estradiol represented 38.9 % of the study population. Statistically significant differences in mean BMD between women with low levels of estradiol and normal levels of estradiol (1.16 vs 1.19 g/cm 2 , P = 0.03) were found. The study population's mean BMI was 26.8 kg/m 2 ; 64.5 % of the population had BMI > 25.0, 43.0 % were overweight, and 21.5 % had some grade of obesity. On average, women with normal levels of estradiol had slightly higher BMI than women with low levels of estradiol (26.9 vs. 26.7 kg/m 2 ). In addition, women with normal levels of estradiol had slightly higher dietary vitamin D intake than women with low levels of estradiol (211 vs. 235 IU/day); however, this difference was not statistically significant.
According to the WHO classification of osteopenia and osteoporosis, the prevalence of premenopausal women with osteopenia was 30.1 %, and the prevalence of osteoporosis was 2.1 % (data not shown). The average age at first occurrence of menstruation in our study population was 12.6 years (SD = 1.5 years), and the average number of gestations was 2.8 (SD = 1.3). The study population's cumulative mean time of breastfeeding was 9.4 months (SD = 10.4 months), and their mean duration of contraceptive use was 6.1 months (SD = 18.0 months). The effects of age-adjusted level of serum estradiol and other risk factors on BMD are shown in [fig_ref] Table 2: Effect of levels of serum estradiol and lifestyle factors on BMD in... [/fig_ref]. In the linear regression analysis levels of estradiol was positively associated with total, lumbar spine and femoral neck BMD (TBMD β = 0.11, P = 0.05, LSBMD β = 0.16, P = 0.01 and FNBMD β = 0.14, P = 0.02). Furthermore, BMI was also positively related to total BMD (β = 0.39, P < 0.001), lumbar spine BMD (β = 0.25, P = 0.001), and with femoral neck BMD (β = 0.40, P < 0.001). Additionally, dietary vitamin D intake (TBMD β = 0.17, P = 0.004, FNBMD β = 0.26, P = 0.01, and LSBMD β = 0.14, P = 0.02), and physical activity (TBMD β = 0.12, P = 0.02, and FNBMD β = 0.15, P = 0.01), were each positively associated with BMD.
In the logistic regression analysis, the odds of low BMD increase significantly when the premenopausal females had low levels of serum estradiol (OR = 4.93; 95 % CI: 2.1, 11. [fig_ref] Table 3: Odds ratio of low bone mineral density according to levels of serum... [/fig_ref]. Further analysis of interaction between smoking status and levels of serum estradiol revealed that the effect of tobacco use increase in the low serum estradiol group (data no shown).
# Discussion
While low bone mineral density (osteopenia/osteoporosis) is considered as a global public health problem, however, its epidemiology in premenopausal women persists unclear. The present study denotes an original contribution to the area of low bone mineral density in premenopausal Mexican females. We found a negative relationship between low levels of serum estradiol and low bone mineral density. Furthermore, we found that not only advancing age, but also tobacco use, and caffeine intake, were determinants of low bone mineral density. On the other hand, vitamin D intake, physical activity, and higher BMI may represent a protective factor, however, BMI has been clearly identified as a cardiovascular and metabolic risk factor.
Our findings are coherent with prior reports, which showed that advancing age was related to small levels of bone mineral density [bib_ref] Measures of bioavailable serum testosterone and estradiol and their relationships with muscle..., Van Geel [/bib_ref] [bib_ref] Bone mineral density, body mass index and cigarette smoking among Irani women:..., Baheiraei [/bib_ref]. According our data, each year rise in age was projected to cross-sectionally decline 0.6 % in lumbar spine (LSBMD) or 0.8 % in femoral neck bone mineral density (FNBMD) (data no show). This estimate is consistent with the previous finding that a decrease of 0.8 % in LSBMD and FNBMD occurs annually [bib_ref] Bone mineral density, body mass index and cigarette smoking among Irani women:..., Baheiraei [/bib_ref]. In addition, we found that females in the oldest age group (46-48 years) had double the risk of lower bone mineral density compared with the youngest study participants (40-42 years) [OR = 2.; 95 % CI: 0.92, 4.13]. BMD (g/cm 2 ) total bone mineral density, TBMD (g/cm 2 ) whole body bone mineral density, LSBMD (g/cm 2 ) lumbar spine bone mineral density, FNBMD femoral neck bone mineral density, BMI body mass index (normal <25.0 kg/m 2 , overweight ≥ 25.0 -<30.0 kg/m 2 , obesity ≥ 30.0 kg/m 2 ) a Coefficients obtained by multiple linear regression analysis BMI appears to be an additional determinant of bone mineral density, as we found that BMI is positively associated with bone mineral density (β = 0.39; p < 0.001). Additionally, we found that overweight females had 56 % less risk of low BMD contrasted with females who had a BMI lower than 25.0 kg/m 2 . This result is consistent with previous findings of a positive relationship between BMI and bone mineral density [bib_ref] Osteoporosis and low bone mass premenopausal and permimenopausal women, Moreira [/bib_ref] [bib_ref] Osteoporosis risk un premenopausal women, Shery [/bib_ref] [bib_ref] Physical activity during life of curse and bone mass, Bielemann [/bib_ref]. BMI's effect on BMD might be determined via blood estrogen levels [bib_ref] Prediction of bone mineral density by age, body mass index and menopausal..., Vaishali [/bib_ref] , and may be explained by peripheral aromatization of androgens to estrogens in fat tissue; however, it does not seems to be the mechanism because we adjusted by estrogen levels in the study.
Unlike the cases of weight and age, the impact of tobacco use on bone mineral density has not been conclusively recognized. We tested how tobacco use effects bone mineral density, or the risk of low bone density, in premenopausal women, and found that current or past cigarette consumption was significantly associated with decreased FNBMD. The odds ratio that a current/past smoker would have low bone mineral density was 1.97 (95 % CI: 1.05, 3.72). However, a meta-analysis suggests that cigarette smoking has no detectable effect on bone mineral density [bib_ref] To drink or not to drink: How are alcohol, caffeine and past..., Ilich [/bib_ref]. This risk increased in women with low estradiol levels, what makes postmenopausal women a higher susceptibility group.
Equal to this paper previous studies had shown a negative association between caffeine (>2 cups/day or 200-300 mg caffeine/day), and BMD in most of the skeletal. The mechanism proposed is by increasing urinary excretion of calcium and a possibly reduced endogenous Ca absorption. This association deleterious effect of caffeine has been noted when dietary calcium is low [bib_ref] Influence of number of pregnancies on bone mineral density in posmenopausal women..., Gur [/bib_ref] [bib_ref] Associations of calcium intake and physical activity with bone density and size..., Uusi [/bib_ref].
Estradiol has been extensively studied as a prognostic factor of bone health among postmenopausal females, although this relationship has not been thoroughly studied in premenopausal women. We found an important association between serum levels of estradiol and total bone mineral density (β = 0.11; P = 0.05). In addition, our data show that subjects with low concentrations of serum estradiol (<60 pg/μL) had 4.93 times greater odds of low bone mineral density compared with subjects who had normal concentrations of serum estradiol (≥60 pg/μL). Estradiol could create positive bone effects through multiple potential pathways; it diminishes initiation of bone metabolic units, it antagonizes parathyroid hormone stimulus of bone reabsorption, it also may improve the survival of osteoblasts via local cytokines or other growth factors, and it enhances the efficiency of gastrointestinal calcium absorption and renal calcium conservation [bib_ref] Effect of endogenous estradiol levels on bone resorption and bone mineral density..., Mastaglia [/bib_ref]. Some or all of these actions may be responsive to low levels of estradiol. Our data support estradiol's effect on the reduction of bone mineral density. While estradiol could indirectly influence bone density through body weight, we adjusted our analysis for body weight and still found a strong association.
The results of the present study must be understood with context of some restrictions. First, we could not measure the peak bone mass, bone turnover markers, which represent the optimal bone mass and function of an individual. In this study, the stage of the menstrual cycle, which could influence the assessment of serum estradiol levels for each woman, was not determined. Though, as suggested in a previous study [bib_ref] Quantification of the relative contribution of estrogen to bone mineral density in..., Ho-Pham [/bib_ref] , the intraindividual variation was measured in the statistical analysis as an element of the random error in the multiple linear regression models. The association we found between levels of serum estradiol and other lifestyle risk factors and bone mineral density cannot be interpreted as causal, because the study was a cross-sectional investigation. This study was carried out in a group of working class, seemingly healthy female workers in a specific site in Mexico. While these women cannot be considered representative of the Mexican female population as a whole, they may be considered representative of middle to low income female adults residing in the urban areas of central Mexico. Furthermore, we only include specific women of the HWCS, which may introduce a selection bias; however, the women included in this study have similar characteristics compared with the females that were not incorporated in our final analysis.
# Conclusions
In summary our results propose that, rising age, body mass index, tobacco use, caffeine and vitamin D consumption, are significant factors of bone mineral density in premenopausal Mexican women. Additionally, our data suggest that estradiol is a significant determinant of total bone mineral density, lumbar spine and femoral neck in premenopausal Mexican females, which is coherent with the theory that estrogen is an important hormone in the maturation, mineralization and maintenance of the bone in females. More prospective investigations are necessary to clarify the clinical implication of premenopausal women circulating estradiol on subsequent fracture risk. Finally, our results can possibly provide evidence in the direction of the elaboration of more effective public policies for the health encouragement and osteoporosis prevention in Mexican female population. . We wish to express our gratitude to everyone who contributed to make this study possible.
## Availability of data and materials
The datasets generated during and/or analyzed during the current study are not publicly available due to protecting participant confidentiality, but are available from the corresponding autor ([email protected]) or with Dr. Jorge Salmerón ([email protected]) on reasonable request.
[table] Table 1: Characteristics of the study population by levels of serum estradiol [/table]
[table] Table 2: Effect of levels of serum estradiol and lifestyle factors on BMD in premenopausal women [/table]
[table] Table 3: Odds ratio of low bone mineral density according to levels of serum estradiol and lifestyle factors Low bone mineral density b OR a (CI 95 %) P-value Odds ratios derived by logistic regression analysis. b Low bone mineral density defined as: (osteoporosis if T-score ≤ −2.5; and osteopenia if −2.5 < T-score ≤ −1.0) [/table]
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Lactational Amenorrhea: Neuroendocrine Pathways Controlling Fertility and Bone Turnover
# Introduction
According to both WHO and American Academy of Pediatrics recommendations [bib_ref] Breastfeeding and the Use of Human Milk, Eidelman [/bib_ref] , every newborn should be breastfed within 1 h of life. Exclusive breastfeeding should continue until the baby is 6 months old. At that age, first complementary foods can be introduced, but breastfeeding continuation is advised up to 2 years of age or beyond. Breastfeeding is associated with various short-term and long-term health benefits for both babies and mothers. Current meta-analysis shows that longer breastfed children are better protected against child infectious diseases and have fewer dental malocclusions and higher intelligence compared to those who are breastfed for shorter periods or not breastfed at all [bib_ref] Breastfeeding in the 21st century: Epidemiology, mechanisms, and lifelong effect, Victora [/bib_ref]. Moreover, breastfeeding reduces mortality from sudden infant death syndrome and necrotizing enterocolitis [bib_ref] Breastfeeding and maternal and infant health outcomes in developed countries, Ip [/bib_ref]. There is also growing evidence that breastfed children may be less likely to suffer from obesity and diabetes in the future as compared to non-breastfed babies [bib_ref] Breastfeeding in the 21st century: Epidemiology, mechanisms, and lifelong effect, Victora [/bib_ref].
It was proved that breastfeeding improves parental sleep duration and has a positive impact on mood and reaction to stress among breastfeeding women, diminishing the chances of postpartum depression development [bib_ref] Differences Between Exclusive Breastfeeders, Formula-Feeders, and Controls: A Study of Stress, Mood,..., Groër [/bib_ref] [bib_ref] The Relationship between Postpartum Depression and Breastfeeding, Hamdan [/bib_ref]. Furthermore, it decreases the risk of breast and ovarian cancers by 26% and 37%, respectively [bib_ref] Breastfeeding and maternal health outcomes: A systematic review and meta-analysis, Chowdhury [/bib_ref]. The risk of type 2 diabetes is also significantly lowered in lactating women [bib_ref] Breastfeeding and the maternal risk of type 2 diabetes: A systematic review..., Aune [/bib_ref]. Satisfaction and continuation of breastfeeding depend on many aspects such as time of delivery, diet consumption, malnutrition, healthy habits, educational level, and employment status [bib_ref] A Review of Bioactive Factors in Human Breastmilk: A Focus on Prematurity, Gila-Diaz [/bib_ref] [bib_ref] Multidimensional Approach to Assess Nutrition and Lifestyle in Breastfeeding Women during the..., Gila-Díaz [/bib_ref].
Amenorrhea is defined as a pause in menstrual bleedings which last more than 6 months during reproductive years. Lactational amenorrhea occurs during postpartum weeks. It prolongs the intervals between following births and thus is related to family history planning.
Our study summarizes the effect that breastfeeding exerts on the hypothalamuspituitary axis and the whole organism, particularly considering fertility changes. Moreover, we discuss the possibility of the use of lactation as contraception, along with this method's prevalence, efficacy, and influencing factors. We also review the literature on the topic of lactational bone loss: its mechanism, severity, and persistence throughout life.
## The physiological aspects of lactation
During pregnancy, the mammary gland undergoes a series of structural and functional changes in order to prepare itself for milk production. A key role in glandular tissue development is played by three hormones: estrogen, progesterone, and prolactin. Estrogen promotes extensive lactiferous ducts development, precisely ductal elongation, while progesterone and prolactin stimulate profuse alveolar-lobular formation [bib_ref] Establishing a framework for the functional mammary gland: From endocrinology to morphology, Hovey [/bib_ref]. Estrogen also stimulates prolactin secretion and increases the number of prolactin receptors in mammary gland cells [bib_ref] Hormones, Mammary Growth, and Lactation: A 41-Year Perspective, Tucker [/bib_ref]. The action of prolactin in the mammary gland is mediated through its receptor, which activates the JAK/STAT signaling pathway for transcription of genes essential in milk synthesis, e.g., casein [bib_ref] Three lactation-related hormones: Regulation of hypothalamus-pituitary axis and function on lactation, Ni [/bib_ref]. However, during pregnancy, high levels of progesterone block the stimulating effect of estrogen on prolactin, consequently inhibiting milk synthesis until the time of delivery when the placenta is removed.
The actual process of breastfeeding is controlled by the let-down reflex [bib_ref] Oxytocin and Brain Plasticity, Froemke [/bib_ref] [bib_ref] Neurochemical Regulation of Oxytocin Secretion in Lactation, Crowley [/bib_ref]. When a baby is suckling a nipple, it stimulates mechanoreceptors located there. Newly created ascending sensory information is transported all the way up via the spinal cord to reach the hypothalamic paraventricular nucleus (PVN) and stimulate oxytocin production [bib_ref] Neuroendocrine Regulation of Lactation and Milk Production, Crowley [/bib_ref]. Oxytocin promotes the contractile activity of myoepithelial cells that surround alveoli. Moreover, oxytocin is the main prolactin-releasing hormone in contrast to dopamine that inhibits prolactin secretion [bib_ref] Three lactation-related hormones: Regulation of hypothalamus-pituitary axis and function on lactation, Ni [/bib_ref]. Prolactin stimulates epithelial alveolar cells to produce milk for the purpose of keeping pace with the baby's needs [bib_ref] Hormonal Regulation of Mammary Differentiation and Milk Secretion, Neville [/bib_ref]. Another neurotransmitter, serotonin, is suggested to be an additional feedback inhibitor of lactation, decreasing milk volume produced [bib_ref] Evaluation of Serotonin as a Feedback Inhibitor of Lactation in the Bovine, Hernandez [/bib_ref].
Metabolism-related hormones are also essential in the process of both milk production and mammary gland development.
Growth hormone (GH), along with prolactin, is essential for milk secretion [bib_ref] Three lactation-related hormones: Regulation of hypothalamus-pituitary axis and function on lactation, Ni [/bib_ref]. It was shown that on the sixth day of lactation, GH on its own can maintain 50% milk production in the absence of PRL, whereas suppression of both GH and PRL can totally eliminate milk secretion [bib_ref] Investigation of the mechanism of action of growth hormone in stimulating lactation..., Flint [/bib_ref]. Because of its ability to increase milk volume, growth hormone administration has been used to enhance milk production in women [bib_ref] Growth Hormone Increases Breast Milk Volumes in Mothers of Preterm Infants, Gunn [/bib_ref]. GH also plays an important role in ductal branching [bib_ref] Establishing a framework for the functional mammary gland: From endocrinology to morphology, Hovey [/bib_ref].
Insulin, together with glucocorticoids, is essential in the structural mammary gland development by regulating tight junction formationand stimulation of cells differentiation [bib_ref] Glucocorticoids Maintain the Extracellular Matrix of Differentiated Mammary Tissue during Explant and..., Casey [/bib_ref]. It was also shown that together with prolactin, they play a key role in the regulation of milk protein synthesis [bib_ref] Insulin, a key regulator of hormone responsive milk protein synthesis during lactogenesis..., Menzies [/bib_ref].
Suckling provides supply in the breast milk and at the same time ensures lactational amenorrhea. Milk is known as the best food for infants. It provides all necessary compounds for a rapidly growing organism. One of the important components is calcium; its adequate amount is supplied mostly by maternal bone turnover. During lactation, bone remodeling is under hormonal stimulation and can affect lactational osteoporosis due to osteoclasts activation. The longer suckling and lactation lasts, the bigger bone loss occurs. This is further discussed in Section 5.
There are also factors that, at first thought, may not be considered as important in the context of lactation, but in fact, they may cause a major disruption of this process. Such conditions include obesity, alcohol consumption, and stress [bib_ref] Reduced dopaminergic tone during lactation is permissive to the hypothalamic stimulus for..., Silva [/bib_ref] [bib_ref] Alcohol and lactation: A systematic review, Giglia [/bib_ref] [bib_ref] Biological underpinnings of breastfeeding challenges: The role of genetics, diet, and environment..., Lee [/bib_ref] [bib_ref] Association of Maternal Obesity Before Conception with Poor Lactation Performance, Rasmussen [/bib_ref] [bib_ref] Prepregnant Overweight and Obesity Diminish the Prolactin Response to Suckling in the..., Rasmussen [/bib_ref] [bib_ref] Obesity impairs lactation performance in mice by inducing prolactin resistance, Buonfiglio [/bib_ref] [bib_ref] Decreased IGF Type 1 Receptor Signaling in Mammary Epithelium during Pregnancy Leads..., Sun [/bib_ref] [bib_ref] Effect of different doses of ethanol on the milk-ejecting reflex in lactating..., Cobo [/bib_ref].
Mother's well-being is crucial for proper breastfeeding, as it was shown that psychological stress or pain decreases milk volume [bib_ref] The let-down reflex in human lactation, Newton [/bib_ref]. It is assumed to be the result of decreased oxytocin release in response to unfavorable conditions. Malnutrition is a factor that stands for smaller milk production and less nutritious milk composition [bib_ref] Lactational amenorrhoea among adolescent girls in low-income and middleincome countries: A systematic..., Figaroa [/bib_ref].
It is estimated that 82% of women who give preterm birth demonstrate delayed secretory activation. It may also happen in case of delivery by cesarean section, maternal obesity, diabetes or gestational diabetes, and primiparity. Delayed secretory activation is associated with an increased risk of early cessation of lactation [bib_ref] What Evidence Do We Have for Pharmaceutical Galactagogues in the Treatment of..., Grzeskowiak [/bib_ref]. In case of insufficient milk production, called lactational insufficiency, hormonal changes may lead to ovulatory cycles and menstrual bleedings, which increases the risk of short intervals between pregnancies.
Mostly, non-pharmacological strategies, including breastfeeding counseling, regular milk removal, dealing with co-morbidities like anemia, diabetes, hypothyroidism, or quitting smoking, are used in such situations. If this strategy is not efficient, substances increasing milk supply, called galactagogues, may provide support. Galactagogues act mainly by stimulation of prolactin and oxytocin. Pharmacological strategy is based on dopamine antagonists-domperidone and metoclopramide-with proven effectiveness in improving milk production. There are limited data available about the use of sulpiride, growth hormone, recombinant human prolactin, thyrotrophin-releasing hormone, oxytocin, or metformin in case of lactational insufficiency [bib_ref] What Evidence Do We Have for Pharmaceutical Galactagogues in the Treatment of..., Grzeskowiak [/bib_ref]. Group of herbal/dietary galactagogues of unsure effectiveness is widely represented by fenugreek, blessed thistle, fennel, milk thistle, ginger, brewer's yeast, lactation cookies, or a combination of herbs. In a study by McBride et al., 60% of Australian women tried some method of lactation support with the highest efficacy while using domperidone [bib_ref] Use and experiences of galactagogues while breastfeeding among Australian women, Mcbride [/bib_ref]. Despite the fact that there are no data about the role of galactagogues in returning to regular bleedings, it can be assumed that by the increase in breast-milk supply, we prolong lactational amenorrhea. More research is needed in this field.
## Endocrine control of lactational amenorrhea
The female hypothalamus-pituitary-ovarian (HPO) axis is significantly altered during both pregnancy and lactation [bib_ref] 60 Years of Neuroendocrinology: The hypothalamo-prolactin axis, Grattan [/bib_ref]. During the pregnancy, placental steroids inhibit gonadotropin-secreting pituitary function, which is reflected by measuring at term LH pituitary content corresponding to 1% of its normal value [bib_ref] Luteinizing Hormone Content of the Pituitary Gland in Pregnant and Non-Pregnant Women, De La Lastra [/bib_ref]. After this period, the suckinginduced disruption of the HPO axis leads to the physiologically appropriate amenorrhea.
Follicle-stimulating hormone (FSH) reaches levels characteristic for the early follicular phase in 4 weeks postpartum, whether or not breastfeeding occurs [bib_ref] Pulsatile secretion of LH in relation to the resumption of ovarian activity..., Glasier [/bib_ref]. The effect of FSH action on follicle development during lactation was observed during an ultrasound exam, which revealed that after 12 weeks postpartum, follicles can reach even 20 mm diameter [bib_ref] Ultrasonographic patterns of ovarian activity during breastfeeding, Flynn [/bib_ref]. It is important to emphasize the fact that these pre-ovulatory-sized follicles showed no or minimal steroidogenic activity due to the inadequate pulsatile LH secretion. Almost throughout the entire period of amenorrhea, until normal menstrual function resumes, the levels of both main FSH-inhibiting hormones (estradiol and inhibin B) remain low, allowing FSH to maintain its concentration in a range typical for the follicular phase [bib_ref] Serum inhibin during lactation: Relation to the gonadotrophins and gonadal steroids, Burger [/bib_ref].
LH plasma concertation may also reach low normal levels within 4 weeks postpartum. However, the disruption in the pulsatile rhythm of LH release seems to block the key events of the menstrual cycle [bib_ref] Physiological Mechanisms Underlying Lactational Amenorrhea, Mcneilly [/bib_ref]. During the follicular phase of the normal menstrual cycle, the frequency of stimulation reaches 1 LH pulse per hour, whereas, in the study including lactating women, the mean number of significant LH pulses over the 24 h at 4 and 8 weeks postpartum was, respectively, 0.56 and 3.36 [bib_ref] The 24 h pattern of pulsatile luteinizing hormone, follicle stimulating hormone and..., Tay [/bib_ref]. As lactation progresses and the duration of daily breastfeeding declines, the frequency of LH secretion increases to reach frequency characteristic for the normal follicular phase, and this is the first step for fertility resumption [bib_ref] Pulsatile secretion of LH in relation to the resumption of ovarian activity..., Glasier [/bib_ref]. It has been shown that breastfeeding at least five times a day with a duration maintained above 65 min maintains the state of amenorrhea [bib_ref] Influence of breast-feeding pattern on pituitary-ovarian axis of women in an industrialized..., Andersen [/bib_ref]. However, the exact mechanism explaining how the frequency and duration of suckling lead to the suppression of LH pulses is still not exactly explained.
Secretion of both gonadotropins is controlled by gonadotropin-releasing hormone (GnRH), produced and released from GnRH neurons located in the hypothalamus [bib_ref] The Neurobiology of Preovulatory and Estradiol-Induced Gonadotropin-Releasing Hormone Surges, Christian-Hinman [/bib_ref]. It is well known that each LH pulse is produced by a corresponding GnRH pulsatile secretion, which means that during the normal follicular phase, GnRH is released once per hour [bib_ref] GnRH Pulsatility, the Pituitary Response and Reproductive Dysfunction, Tsutsumi [/bib_ref]. The release of FSH occurs much less often than GnRH pulses, and low GnRH pulse frequency tends to favor FSH production [bib_ref] The GPR54 Gene as a Regulator of Puberty, Seminara [/bib_ref].
According to current knowledge, neuropeptide kisspeptin (Kiss1) is considered as a primary gatekeeper of the HPO axis action, as it is the most potent stimulator of GnRH secretion yet identified [bib_ref] The role of changing pulse frequency in the regulation of ovulation, Marshall [/bib_ref] [bib_ref] Activation of Gonadotropin-Releasing Hormone Neurons by Kisspeptin as a Neuroendocrine Switch for..., Han [/bib_ref]. Kisspeptins are encoded by the Kiss1 gene and act through their receptors encoded by the Kiss1R gene, which is expressed in the majority of GnRH neurons [bib_ref] Activation of Gonadotropin-Releasing Hormone Neurons by Kisspeptin as a Neuroendocrine Switch for..., Han [/bib_ref] [bib_ref] Kisspeptin directly stimulates gonadotropin-releasing hormone release via G protein-coupled receptor 54, Messager [/bib_ref]. Mutation in either of these genes is linked to idiopathic hypothalamic hypogonadism and low amplitude of LH pulses in humans, which underscores the role of kisspeptin in the proper function of the HPO axis [bib_ref] The GPR54 Gene as a Regulator of Puberty, Seminara [/bib_ref] [bib_ref] Hypogonadotropic hypogonadism due to loss of function of the KiSS1-derived peptide receptor..., De Roux [/bib_ref]. In rodents, kisspeptin neurons are located in two regions: the arcuate nucleus (ARC) and the anteroventral periventricular nucleus (AVPV), which are involved in GnRH/LH pulsatile secretion and pre-ovulatory LH surge generation, respectively [bib_ref] Kiss1 Neurons in the Forebrain as Central Processors for Generating the Preovulatory..., Smith [/bib_ref] [bib_ref] Neuropeptidergic modulation of GnRH neuronal activity and GnRH secretion controlling reproduction: Insights..., Spergel [/bib_ref]. ARC neurons, in addition to kisspeptin, also synthesize and release two co-neuropeptides-neurokinin B (NKB) and dynorphin (DYN) [bib_ref] The neuroendocrine basis of lactation-induced suppression of GnRH: Role of kisspeptin and..., Smith [/bib_ref]. Neurons that co-express Kiss1, NKB, and DYN are known as KNDy neurons, and current evidence strongly suggests that KNDy cells, as well as KNDy neuropeptides, play an important role in the control of fertility by influencing GnRH activity by acting on their cell bodies and their secretory terminals [bib_ref] Kisspeptin/Neurokinin B/Dynorphin (KNDy) Cells of the Arcuate Nucleus: A Central Node in..., Lehman [/bib_ref].
The suppression of Kiss1 during lactation may be the key factor in the suppression of GnRH [bib_ref] The neuroendocrine basis of lactation-induced suppression of GnRH: Role of kisspeptin and..., Smith [/bib_ref]. During lactation in the rat, Kiss 1 mRNA expression is greatly reduced both in the ARC and AVPV [bib_ref] Inhibition of Metastin (Kisspeptin-54)-GPR54 Signaling in the Arcuate Nucleus-Median Eminence Region during..., Yamada [/bib_ref]. Furthermore, in the ARC KNDy cells, NKB mRNA is also significantly inhibited [bib_ref] Characterisation of Arcuate Nucleus Kisspeptin/Neurokinin B Neuronal Projections and Regulation during Lactation..., True [/bib_ref]. In addition, central administration of Kiss1 during lactation results in increased LH secretion [bib_ref] Inhibition of Metastin (Kisspeptin-54)-GPR54 Signaling in the Arcuate Nucleus-Median Eminence Region during..., Yamada [/bib_ref] , whereas microinjections of selective kisspeptin antagonists into the ARC profoundly suppress pulsatile LH secretion in rats [bib_ref] Kisspeptin Signalling in the Hypothalamic Arcuate Nucleus Regulates GnRH Pulse Generator Frequency..., Li [/bib_ref].
The reduction in Kiss1 during amenorrhea is suggested to be linked to the occurring in the physical lactation state of hyperprolactinemia [bib_ref] Hyperprolactinemia Alters the Frequency and Amplitude of Pulsatile Luteinizing Hormone Secretion in..., Cohen-Becker [/bib_ref] [bib_ref] Suppression of Pulsatile LH Secretion, Pituitary GnRH Receptor Content and Pituitary Responsiveness..., Fox [/bib_ref]. A study by Kotani et al. reveals that plasma Kiss1 levels are not totally reduced in lactational amenorrhea, which may suggest that the role of the serum kisspeptin differs from those performed in the brain [bib_ref] Plasma kisspeptin levels in lactational amenorrhea, Kotani [/bib_ref].
It was shown that infusion of prolactin in female mice suppressed ovarian function by inhibition of GnRH and Kiss1 mRNA expression, while kisspeptin administration restored normal ovarian function [bib_ref] Hyperprolactinemia-induced ovarian acyclicity is reversed by kisspeptin administration, Sonigo [/bib_ref]. Research on rats has also shown that PRL acts on ARC neurons to inhibit kisspeptin expression [bib_ref] Prolactin Regulates Kisspeptin Neurons in the Arcuate Nucleus to Suppress LH Secretion..., Araujo-Lopes [/bib_ref] [bib_ref] Kisspeptin Signaling in the Brain, Oakley [/bib_ref]. Presented data advocate the role of Kiss1 neurons in the mediation of prolactin's inhibitory effect on GnRH release [fig_ref] Figure 1: Neuroendocrine control of amenorrhea [/fig_ref]. ARC neurons to inhibit kisspeptin expression [bib_ref] Prolactin Regulates Kisspeptin Neurons in the Arcuate Nucleus to Suppress LH Secretion..., Araujo-Lopes [/bib_ref] [bib_ref] Kisspeptin Signaling in the Brain, Oakley [/bib_ref]. Presented data advocate the role of Kiss1 neurons in the mediation of prolactin's inhibitory effect on GnRH release [fig_ref] Figure 1: Neuroendocrine control of amenorrhea [/fig_ref]. Even though exact mechanisms standing behind hypothalamus-pituitary-ovarian axis alternation during lactation are still not clear, a general overview of events leading to amenorrhea may be suggested. Suckling remains the most important stimulus maintaining suppressive effect on ovaries after pregnancy. Breastfeeding is accompanied by high levels of prolactin that remain higher than normal until the frequency and duration of daily suckling will be this low to let normal menstrual function resume. Hyperprolactinemia induces the suppression of hypothalamic Kiss1 neurons that directly control the pulsatile release of GnRH. Disruption in the pulsatile manner of GnRH secretion results in a strongly decreased frequency of corresponding LH pulses. Inadequate LH secretion and lack of pre-ovulatory surge inhibit the progression of the follicular phase of a menstrual cycle resulting in anovulation and amenorrhea [bib_ref] Hypothalamic-Pituitary-Ovarian Axis Reactivation by Kisspeptin-10 in Hyperprolactinemic Women with Chronic Amenorrhea, Millar [/bib_ref].
## Lactational amenorrhea as natural contraceptive method
The lactational amenorrhea method (LAM) is one of the family planning methods based on the natural protection of breastfeeding against pregnancy [bib_ref] Lactational amenorrhoea method for family planning, Van Der Wijden [/bib_ref].
It is commonly known that during the lactation period, there is a decline in the woman's fertility, but the exact conditions must be fulfilled to fully exploit the potential of breastfeeding as a method of contraception [bib_ref] Contraceptive use during lactational amenorrhea, Hight-Laukaran [/bib_ref].
These conditions were first introduced during the Bellagio Consensus Conference in Italy in 1988 when an international group of scientists gathered to establish the safe and effective use of the lactational amenorrhea method: 1. A period of sixth months after delivery; Even though exact mechanisms standing behind hypothalamus-pituitary-ovarian axis alternation during lactation are still not clear, a general overview of events leading to amenorrhea may be suggested. Suckling remains the most important stimulus maintaining suppressive effect on ovaries after pregnancy. Breastfeeding is accompanied by high levels of prolactin that remain higher than normal until the frequency and duration of daily suckling will be this low to let normal menstrual function resume. Hyperprolactinemia induces the suppression of hypothalamic Kiss1 neurons that directly control the pulsatile release of GnRH. Disruption in the pulsatile manner of GnRH secretion results in a strongly decreased frequency of corresponding LH pulses. Inadequate LH secretion and lack of pre-ovulatory surge inhibit the progression of the follicular phase of a menstrual cycle resulting in anovulation and amenorrhea [bib_ref] Hypothalamic-Pituitary-Ovarian Axis Reactivation by Kisspeptin-10 in Hyperprolactinemic Women with Chronic Amenorrhea, Millar [/bib_ref].
## Lactational amenorrhea as natural contraceptive method
The lactational amenorrhea method (LAM) is one of the family planning methods based on the natural protection of breastfeeding against pregnancy [bib_ref] Lactational amenorrhoea method for family planning, Van Der Wijden [/bib_ref].
It is commonly known that during the lactation period, there is a decline in the woman's fertility, but the exact conditions must be fulfilled to fully exploit the potential of breastfeeding as a method of contraception [bib_ref] Contraceptive use during lactational amenorrhea, Hight-Laukaran [/bib_ref].
These conditions were first introduced during the Bellagio Consensus Conference in Italy in 1988 when an international group of scientists gathered to establish the safe and effective use of the lactational amenorrhea method:
## 1.
A period of sixth months after delivery; 2.
"Full" or "nearly full" breastfeeding; 3.
Postpartum amenorrhea.
In that way, women can achieve 98% protection against pregnancy in the first six postpartum months [bib_ref] First ovulation after childbirth: The effect of breast-feeding, Perez [/bib_ref].
When any of the above requirements is no longer met, the risk of conception increases, and therefore, another family planning method should be considered to prevent unwanted pregnancy. If no other contraception form is available, women can eventually benefit from breastfeeding alone to maximize the birth interval [bib_ref] First ovulation after childbirth: The effect of breast-feeding, Perez [/bib_ref].
The effectiveness of LAM was thoroughly examined and confirmed in a number of papers. One of the earliest studies, conducted even before the Bellagio Consensus Conference by Perez et al., clearly illustrated the relationship between the intensity and duration of breastfeeding and the time of ovulation return [bib_ref] First ovulation after childbirth: The effect of breast-feeding, Perez [/bib_ref]. Fewer breastfeeds are significantly connected with ovulating before six months postpartum [bib_ref] Characteristics and determinants of postpartum ovarian function in women in the United..., Campbell [/bib_ref] [bib_ref] Risk of ovulation during lactation, Gray [/bib_ref] , and a similar association is seen in the menses return [bib_ref] Characteristics and determinants of postpartum ovarian function in women in the United..., Campbell [/bib_ref] [bib_ref] Actual use of the lactational amenorrhoea method, Romero-Gutiérrez [/bib_ref]. Some data suggest infant sex correlation with the time of menstrual bleedings' return with the longer duration with males, mostly seen in malnourished, low energy budget societies. Sons seem to be costlier than daughters [bib_ref] Postpartum amenorrhea duration by sex of the newborn in two natural fertility..., Hoi [/bib_ref] , although more research is needed to explain this issue.
The lowest ovulation risk and, consequently, the pregnancy rate was obtained during the first six postpartum months [bib_ref] Risk of ovulation during lactation, Gray [/bib_ref]. Therefore, this period was included in the recommendations.
When first menstruation appears during the first six months after the delivery, it is more likely to be anovular than occurring after that time [bib_ref] Characteristics and determinants of postpartum ovarian function in women in the United..., Campbell [/bib_ref]. The chances of ovulation during that time are from 20% to 45%, according to different studies [bib_ref] Lactational amenorrhoea method for family planning, Van Der Wijden [/bib_ref] [bib_ref] Risk of ovulation during lactation, Gray [/bib_ref] [bib_ref] Postpartum Sexuality and the Lactational Amenorrhea Method for Contraception, Labbok [/bib_ref]. Furthermore, the percentage of normal, physiologic ovulations rises from 47% during the first six months to 76% after [bib_ref] Risk of ovulation during lactation, Gray [/bib_ref]. The six-month time point is also associated with the appearance of the baby's first tooth and, therefore, is often connected with supplementary feeding implementation [bib_ref] Postpartum contraception: The lactational amenorrhea method, Vekemans [/bib_ref].
The term "full or nearly full breastfeeding" means that the baby is given only breastmilk without any other solid or liquid food, or eventually some vitamins, water, juice, or ritualistic feeds in a sporadic way. The introduction of supplementary food causes an abrupt decline in suckling frequency and duration, increasing the risk of ovulation return [bib_ref] Effect of breast-feeding patterns on human birth intervals, Howie [/bib_ref]. During the first six months, the ovulation risk of not menstruating women lowers from about 10% when partial to 1-5% with exclusive breastfeeding [bib_ref] Risk of ovulation during lactation, Gray [/bib_ref] [bib_ref] Postpartum Sexuality and the Lactational Amenorrhea Method for Contraception, Labbok [/bib_ref]. However, some studies proved the high efficacy of LAM after excluding the "exclusive breastfeeding" criterium and stated that in this way, the method would be available for more women [bib_ref] Contraceptive efficacy of lactational amenorrhoea, Kennedy [/bib_ref].
The pregnancy risk increases significantly with the menses return [bib_ref] Interventions to Improve Postpartum Family Planning in Low-and Middle-Income Countries: Program Implications..., Cleland [/bib_ref] , so to avoid unwanted pregnancy, it is essential to immediately implement another form of contraception [bib_ref] Contraceptive efficacy of lactational amenorrhoea, Kennedy [/bib_ref]. Even though first menstrual bleeding is not equal to the complete recovery of fertility, it still gives some information about ovarian activity. In countries where contraception is more difficult to get, it is better to use this amenorrheal period to plan and arrange another contraceptive method than to wait first for the end of the amenorrhea [bib_ref] Lactational amenorrhoea method for family planning, Van Der Wijden [/bib_ref] [bib_ref] Postpartum fertility behaviours and contraceptive use among women in rural Ghana, Eliason [/bib_ref].
Nonhormonal methods of contraception are the best choice while breastfeeding. Women may also consider permanent methods [bib_ref] Contraception and Breastfeeding, Holder [/bib_ref] [bib_ref] The Academy of Breastfeeding Medicine ABM Clinical Protocol #13: Contraception during Breastfeeding,..., Berens [/bib_ref]. Hormonal methods are possible, but their effect and breastfeeding patterns are still under examination [bib_ref] Contraception and Breastfeeding, Holder [/bib_ref]. Research shows that after using LAM, a high percentage of women switch to another method of family planning [bib_ref] Multicenter study of the lactational amenorrhea method (LAM) III: Effectiveness, duration, and..., Peterson [/bib_ref]. The fact that most women tend to resume their sexual activity between 6 and 12 weeks after the delivery only confirms the significance of choosing the most effective contraception method [bib_ref] Postpartum Sexuality and the Lactational Amenorrhea Method for Contraception, Labbok [/bib_ref] [bib_ref] Effects of pregnancy and childbirth on postpartum sexual function: A longitudinal prospective..., Connolly [/bib_ref] [bib_ref] Choice of contraception at 6-8 weeks postpartum in south-eastern Hungary, Vanya [/bib_ref].
Despite the initially established criteria, further research on this topic introduced a couple of factors that might contribute to LAM effectiveness.
Suckling characteristics such as duration and frequency are the most influential factors, and many studies confirmed their significance [bib_ref] Characteristics and determinants of postpartum ovarian function in women in the United..., Campbell [/bib_ref] [bib_ref] Risk of ovulation during lactation, Gray [/bib_ref] [bib_ref] Effect of breast-feeding patterns on human birth intervals, Howie [/bib_ref]. The minimum suckling frequency needed to suppress ovarian activity is at least five times per day, with a duration of no less than 10 min per feeding. Overall daily suckling time should be not less than 65 min. When the parameters are below these values, there is a higher probability of the ovulational cycle occurring [bib_ref] Influence of breast-feeding pattern on pituitary-ovarian axis of women in an industrialized..., Andersen [/bib_ref]. According to Andersen et al., a frequency of from six to seven feedings is sufficient to maintain anovulation while using supplementary feeding less than once per day [bib_ref] Influence of breast-feeding pattern on pituitary-ovarian axis of women in an industrialized..., Andersen [/bib_ref]. Campbell et al.'s study noticed the importance of median feedings interval [bib_ref] Characteristics and determinants of postpartum ovarian function in women in the United..., Campbell [/bib_ref] , which might be directly related to the need for night feed maintenance [bib_ref] Effect of breast-feeding patterns on human birth intervals, Howie [/bib_ref]. Among the supplementary feedings, only the number of bottle feedings was directly related to the frequency of breastfeeding, therefore contributing to the higher risk of ovulation [bib_ref] Characteristics and determinants of postpartum ovarian function in women in the United..., Campbell [/bib_ref].
The way how suckling is discontinued also appears important. Rapid cessation is more likely to be connected with a fertile luteal phase than more gradual weaning [bib_ref] Influence of breast-feeding pattern on pituitary-ovarian axis of women in an industrialized..., Andersen [/bib_ref]. It is presumed that the time of supplementation introduction into the feeding schedule has little influence on LAM efficacy. The level of maternal nutrition also seems to be less significant than suckling behavior [bib_ref] Influence of breast-feeding pattern on pituitary-ovarian axis of women in an industrialized..., Andersen [/bib_ref]. The separation from the baby has a negative impact on LAM. Therefore, the method might not be suitable for working women [bib_ref] The efficacy of the lactational amenorrhea method (LAM) among working women, Valdés [/bib_ref].
Another matter of extreme importance is the acceptance and correctness of the LAM usage among breastfeeding women. Many scientific studies focused on that problem were conducted, and their results vary in different populations [fig_ref] Table 1: Summary of LAM acceptance and correctness in selected countries [/fig_ref]. Legend: "-"-no data; LAM-lactational amenorrhea method.
The results express the need to provide more correct information about natural family planning, including the LAM method during contraception counseling [bib_ref] Knowledge, attitude and practice of natural family planning methods in a population..., Audu [/bib_ref]. Among some populations, one consultation is not enough, and women need to be reminded of LAM efficacy and advantages throughout the postpartum period [bib_ref] Actual use of the lactational amenorrhoea method, Romero-Gutiérrez [/bib_ref]. There are societies in which partners tend not to approve of any kind of family planning. In this case, women's education might be more beneficial if conducted without men's presence [bib_ref] Lactational Amenorrhea Method as a Contraceptive Strategy in Niger. Matern, Sipsma [/bib_ref]. In India, lactational amenorrhea tends to be a reason not to use any other contraceptive method for 8% of women [bib_ref] Knowledge and associated factors of lactational amenorrhea as a contraception method among..., Abraha [/bib_ref]. Applying effective anticonception in the postpartum period is of extreme importance to both mother and child. According to the World Health Organization, the suggested time before the next pregnancy is at least 24 months. The Conde-Agudelo et al.'s retrospective cross-sectional study shows that when pregnancy occurs earlier than six months after the previous one, there is a higher risk of maternal death, bleeding in the third semester, premature membrane rupture, puerperal endometriosis, and anemia [bib_ref] Maternal morbidity and mortality associated with interpregnancy interval: Cross sectional study, Conde-Agudelo [/bib_ref].
To summarize, lactational amenorrhea method might be effectively used as a contraception method, but only among populations with adequate access to health services or another source of reliable and correct information of its use. Strict adherence to the established criteria increases the safety and reliability of LAM and protects against detrimental health consequences of a too short inter-birth interval.
## The lactational bone loss
Another important process occurring during lactational amenorrhea is the change in bone mineral density (BMD). Pregnancy and lactation bring changes in bone turnover because of the need for building material for the development of the fetal skeleton. There is a significant calcium loss during the lactation period with the breastmilk of 200 mg/day on average, but it can vary even among exclusively breastfeeding women [bib_ref] Calcium in Pregnancy and Lactation, Prentice [/bib_ref]. WHO recommends an extra dose of dietary calcium supplementation during pregnancy.
After the delivery, the rapid change in hormonal status takes place. Induction of lactation is associated with the decline of estrogen and progesterone levels [bib_ref] Control of mineral homeostasis during lactation: Interrelationships of 25-hydroxyvitamin D, 24,25-dihydroxyvitamin D,..., Hillman [/bib_ref]. According to Hillman et al.'s study, the protective effect of estrogens on bones is reduced, and the subsequent lactation period cause increased bone remodeling [bib_ref] Control of mineral homeostasis during lactation: Interrelationships of 25-hydroxyvitamin D, 24,25-dihydroxyvitamin D,..., Hillman [/bib_ref]. The lack of estrogens is believed to be one of the main causes of lactational bone loss [bib_ref] Bone loss, contraception and lactation, Mehta [/bib_ref]. During the late lactation period and after the discontinuation of breastfeeding, estrogens return [bib_ref] Control of mineral homeostasis during lactation: Interrelationships of 25-hydroxyvitamin D, 24,25-dihydroxyvitamin D,..., Hillman [/bib_ref] and cause the formation of the bones [bib_ref] Changes in bone mineral density and markers of bone remodeling during lactation..., Cross [/bib_ref]. The state of elevated prolactin level, which occurs during lactation, is also associated with the reduced bone mineral content [bib_ref] Vertebral Body Bone Mineral Content in Hyperprolactinemic Women, Koppelman [/bib_ref].
Bone remodeling involves perforating of trabeculae, a decrease in their number, and an increase in cortical porosity. Physical changes occurring during pregnancy and lactation in women such as weight gain, lack of physical activity, and excessive lumbar lordosis play a role in bone and joint loading. In the aim to provide calcium requirements to the fetus or a newborn, the mother's organism must adapt. During pregnancy, calcium demands are mostly gathered by increased intestinal absorption, while during lactation, calcium homeostasis is gained by increased calcium resorption from bones as a result of the stimulation of osteoclastogenesis and osteoclast-dependent bone turnover [bib_ref] Pregnancy and lactation, a challenge for the skeleton, Winter [/bib_ref]. Osteocytes remodel their perilacunar space, which results in enlargement of lacunae and resorption of the perilacunar matrix. Those changes lead to calcium mobilization from the skeleton [bib_ref] Cathepsin K-deficient osteocytes prevent lactation-induced bone loss and parathyroid hormone suppression, Lotinun [/bib_ref] and temporary bone mineral loss [bib_ref] Maternal Mineral and Bone Metabolism During Pregnancy, Lactation, and Post-Weaning Recovery, Kovacs [/bib_ref].
Due to the lack of estrogen and increased calcium resorption from bones, prolonged lactational amenorrhea can be a potentially threatening condition. Although pregnancyand lactation-induced osteoporosis (PLO) is a rare disease, in specific conditions like rheumatic diseases, glucocorticosteroids, or oncology treatments, osteoporosis with bone fractures may occur [bib_ref] Pregnancy and lactation, a challenge for the skeleton, Winter [/bib_ref].
A couple of factors influence skeleton demineralization during the lactation period. The most important regulators of calcium metabolism in mammals are parathormone (PTH), parathyroid hormone-related protein (PTHrP), vitamin D metabolites, and prolactin. They increase serum calcium levels mostly by bone resorption. Although PTH is one of the most important hormones regulating calcium metabolism in rodents during lactation, its levels were found to be lower [bib_ref] Changes in bone mineral density and markers of bone remodeling during lactation..., Cross [/bib_ref] or normal [bib_ref] Control of mineral homeostasis during lactation: Interrelationships of 25-hydroxyvitamin D, 24,25-dihydroxyvitamin D,..., Hillman [/bib_ref] in breastfeeding women.
It was proved that mammary cells during lactation produce circulating PTHrP, which might contribute to skeleton resorption [bib_ref] Mammary-specific deletion of parathyroid hormone-related protein preserves bone mass during lactation, Vanhouten [/bib_ref]. PTHrP suppresses and replaces PTH and, in a lack of estrogen condition, stimulates osteoclasts activity.
1,25(OH) 2 D (Calcitriol) level was found to be within normal limits in breastfeeding women [bib_ref] Control of mineral homeostasis during lactation: Interrelationships of 25-hydroxyvitamin D, 24,25-dihydroxyvitamin D,..., Hillman [/bib_ref]. During pregnancy, calcitriol's levels are elevated, stimulating increased calcium intestinal absorption and decreasing its urinary waste. Calcitriol remains elevated even after pregnancy in rodents, while it drops after delivery in humans. Both PTH and calcitriol levels rise during the postweaning period compared to lactation [bib_ref] Changes in bone mineral density and markers of bone remodeling during lactation..., Cross [/bib_ref].
Calcitonin seems to play a protective role during lactation for mothers' skeleton [bib_ref] Maternal Mineral and Bone Metabolism During Pregnancy, Lactation, and Post-Weaning Recovery, Kovacs [/bib_ref].
To summarize, the main factors that cause bones demineralization during lactation in women are PTHrP secreted by mammary tissue and low estrogens levels, while PTH and calcitriol seem not to play a significant role in this process [bib_ref] Maternal Mineral and Bone Metabolism During Pregnancy, Lactation, and Post-Weaning Recovery, Kovacs [/bib_ref].
Cathepsin K (Ctsk) is a cysteine protease produced by osteoclasts. It is a primary enzyme mediating the degradation of the demineralized bone matrix. Osteocytes' expression of Ctsk genes is elevated during lactation. Ctsk regulates osteocytes expression affecting perilacunar resorption and enlargement of osteocyte lacunae. In addition to the induction of osteocytes, Ctsk also stimulates the induction of osteoclasts. A study by Lotinun et al. on a mouse model reveals that deletion of Ctsk decreases perilacunar resorption by osteocytes but also decreases induction of osteoclasts number and their role in bone formation, which normally occurs during lactation in mice [bib_ref] Cathepsin K-deficient osteocytes prevent lactation-induced bone loss and parathyroid hormone suppression, Lotinun [/bib_ref].
During lactation, the loss of bone mineral density is particularly visible at trabecularrich sites such as lumbar spine bone [bib_ref] Changes in bone mineral density and markers of bone remodeling during lactation..., Cross [/bib_ref] [bib_ref] Bone Mineral Changes during Pregnancy and Lactation: A Longitudinal Cohort Study, Kolthoff [/bib_ref] [bib_ref] Bone changes after 3 mo of lactation: Influence of calcium intake, breast-milk..., Laskey [/bib_ref] [bib_ref] Changes in bone mineral density and calcium metabolism in breastfeeding women: A..., Affinito [/bib_ref] [bib_ref] The effects of lactation on bone mineral content in healthy postpartum women, Hayslip [/bib_ref] [bib_ref] Osteoporosis in Premenopausal Women: A Clinical Narrative Review by the ECTS and..., Pepe [/bib_ref] and femoral neck [bib_ref] Bone Mineral Changes during Pregnancy and Lactation: A Longitudinal Cohort Study, Kolthoff [/bib_ref] [bib_ref] Bone changes after 3 mo of lactation: Influence of calcium intake, breast-milk..., Laskey [/bib_ref]. On the other hand, no loss in radius during the 6-month lactation period was observed [bib_ref] Bone Mineral Changes during Pregnancy and Lactation: A Longitudinal Cohort Study, Kolthoff [/bib_ref] [bib_ref] The effects of lactation on bone mineral content in healthy postpartum women, Hayslip [/bib_ref]. However, when comparing 30-35-year-old women with the only significant difference between them being the mean lactation duration, radial bone mass at ultra-distal and midshaft sites was lower among the women that breastfed for a longer period [bib_ref] Osteoporosis in Premenopausal Women: A Clinical Narrative Review by the ECTS and..., Pepe [/bib_ref] [bib_ref] The effect of lactation on peak adult shaft and ultra-distal forearm bone..., Wardlaw [/bib_ref].
Bone resorption can be measured by dual-energy X-ray absorptiometry (DEXA) with 4% changes in spine bone mineral content seen just after 3 months of breastfeeding [bib_ref] Bone changes after 3 mo of lactation: Influence of calcium intake, breast-milk..., Laskey [/bib_ref]. Atkinson et al. measured the mineral loss in the lower femoral shaft and found the average rate of loss in the bone index of 2.2% during 100 days of lactation [bib_ref] Loss of skeletal calcium in lactating women, Atkinson [/bib_ref]. According to Sowers et al., at six months postpartum, breastfeeding women had losses in BMD of the lumbar spine and femoral neck compared to initial values. Hayslip et al. compared exclusively breastfeeding women with the formula-feeding ones, and at six months postpartum, the loss of lumbar spine bone mineral content was associated with breastfeeding, while no significant change happened in the second group [bib_ref] The effects of lactation on bone mineral content in healthy postpartum women, Hayslip [/bib_ref]. Such comparison was also conducted by Affinito et al. In this study, after six months, both lumbar and radial BMD decreased, again with no significant change in a group of women with bromocriptine inhibited lactation [bib_ref] Changes in bone mineral density and calcium metabolism in breastfeeding women: A..., Affinito [/bib_ref] [fig_ref] Table 2: Decrease in BMD depending on the site of measurements and the duration... [/fig_ref]. It seems that the duration of breastfeeding, the volume of produced breastmilk, and the time of returning to regular menses may relate to the magnitude of the changes in bone mineralization. The loss in bone mass was proved to be independent of calcium intake, weight change [bib_ref] Bone Mineral Changes during Pregnancy and Lactation: A Longitudinal Cohort Study, Kolthoff [/bib_ref] [bib_ref] Bone changes after 3 mo of lactation: Influence of calcium intake, breast-milk..., Laskey [/bib_ref] , breast-milk calcium concentration, vitamin D-receptor genotype, or use of the progesterone-only contraceptive pill [bib_ref] Bone changes after 3 mo of lactation: Influence of calcium intake, breast-milk..., Laskey [/bib_ref]. Therefore, hormonal variations in the postpartum period might be the main trigger of skeleton changes [bib_ref] Bone Mineral Changes during Pregnancy and Lactation: A Longitudinal Cohort Study, Kolthoff [/bib_ref].
An interesting observation is that BMD recovery occurs after the cessation of lactation [bib_ref] Bone Mineral Changes during Pregnancy and Lactation: A Longitudinal Cohort Study, Kolthoff [/bib_ref] [bib_ref] Changes in bone mineral density and calcium metabolism in breastfeeding women: A..., Affinito [/bib_ref]. Lamke et al. proved that mothers whose breastfeeding period was shorter than three months first lost and then regained bone mineral content, but there were no losses among those breastfeeding longer [bib_ref] Changes of Bone Mineral Content During Pregnancy and Lactation, Lamke [/bib_ref]. showed no decline in bone density if feeding was shorter than 4 months. Longitudinal studies demonstrated an increase in areal bone mineral density (aBMD) seen after cessation of lactation independently of the duration of breastfeeding. However, the same study reveals a decrease in volumetric bone mineral density (vBMD) depending on the duration of feeding, being still important 18 months postpartum for women feeding longer than 9 months [bib_ref] Changes in Cortical Volumetric Bone Mineral Density and Thickness, and Trabecular Thickness..., Brembeck [/bib_ref]. In another study by Bjørnerem et al., lactational changes were irreversible, despite follow-up of 2.6 years post breastfeeding and 3 years post lactational amenorrhea. Residual deficits remained relative to the beginning of breastfeeding with higher cortical porosity, fewer trabeculae, and lower matrix mineralization [bib_ref] Irreversible Deterioration of Cortical and Trabecular Microstructure Associated with Breastfeeding, Bjørnerem [/bib_ref].
A retrospective analysis of 586 postmenopausal women by Yazici et al. showed that lactation length is not an independent risk factor for low femur BMD or low spine BMD, and changes in bone metabolism during lactation had no effect on postmenopausal BMD measured by DXA [bib_ref] The effect of breast-feeding duration on bone mineral density in postmenopausal Turkish..., Yazici [/bib_ref]. Fox et al., in their study, came to a similar conclusion about radius BMD [bib_ref] Reproductive correlates of bone mass in elderly women. Study of Osteoporotic Fractures..., Fox [/bib_ref]. A prospective observational study among about 6500 women for 16 years showed no associations between parity and duration of feeding with fractures and spine density [bib_ref] Parity and lactation are not associated with incident fragility fractures or radiographic..., Cooke-Hubley [/bib_ref]. Long-term observation reveals possible small, site-specific benefits of parenting and breastfeeding to bone density. The breastfeeding duration of above two years has no significant impact on the postmenopausal fracture risk in most studies [bib_ref] Reproductive history and postmenopausal risk of hip and forearm fracture, Alderman [/bib_ref] , although, in one study, the negative association between lactation and hip fracture risk was noticed [bib_ref] An epidemiologic study of hip fracture in postmenopausal women, Kreiger [/bib_ref].
# Conclusions
Lactation is strictly controlled by a series of reproductive and metabolic hormones that influence both mammary gland development and milk synthesis. Despite the fact that exact mechanisms standing behind hypothalamus-pituitary-ovarian axis alternation during lactation are still not clear, a general overview of events leading to amenorrhea may be suggested. Suckling remains the most important stimulus maintaining suppressive effect on ovaries after pregnancy. Breastfeeding is accompanied by high levels of prolactin that remain higher than normal until the frequency and duration of daily suckling decreases and allows normal menstrual function resumption. Hyperprolactinemia induces the suppression of hypothalamic Kiss1 neurons that directly control the pulsatile release of GnRH. Disruption in the pulsatile manner of GnRH secretion results in a strongly decreased frequency of corresponding LH pulses. Inadequate LH secretion and lack of pre-ovulatory surge inhibits the progression of the follicular phase of a menstrual cycle and results in anovulation and amenorrhea.
The same mechanisms of lactational amenorrhea or anestrus are typical for mammals. Some changes between species may be seen, depending mostly on a suckling or lactating status of an animal, but also factors such as the amount of feeding before and after parturition, level of milk yield, age of the animal, calving difficulty, presence of a bull in the herd, season and its photoperiodism can influence the duration of anestrus in cows and mares [bib_ref] Endocrine regulation of postpartum ovarian activity in cattle: A review, Hanzen [/bib_ref] [bib_ref] Serum Progesterone Levels in Mares in Winter and during Transitional Periods, Koskinen [/bib_ref] [bib_ref] Review: Treatment of Cows with an Extended Postpartum Anestrous Interval, Rhodes [/bib_ref]. The nutritional deficit becomes relatively more important during the third-and fourth-week post-parturition [bib_ref] Endocrine bases of lactational anoestrus in the sow, Quesnel [/bib_ref]. Interestingly, a unique reproductive strategy is represented by swamp wallabies, where females are continuously pregnant and lactating at the same time throughout their reproductive life [bib_ref] Unique reproductive strategy in the swamp wallaby, Menzies [/bib_ref].
If all the established conditions of its use are fulfilled, the lactational amenorrhea method efficiently protects against pregnancy. Because of its accessibility and lack of additional associated costs, LAM might be especially beneficial in low-income, developing countries, where modern contraception is hard to obtain.
LAM is based on the natural and physiologic mechanism of breastfeeding, which allows for its use among religious and ethnic groups. The advantages of breastfeeding for both mother and child and the importance of maintaining the recommended birth interval should be widely spread. Breastfeeding alone is not equal to the LAM method, and therefore, it is not enough to successfully protect against conception. That is why LAM promotion should primarily focus on conditions under which its use is safe and effective.
More studies on larger study groups should be conducted to determine and confirm the impact of behavioral factors, such as suckling parameters, on the LAM efficacy.
Lactational bone loss is a physiologic mechanism that enables providing a sufficient amount of calcium to the newborn. Despite the decline in bone mass during breastfeeding, it rebuilds after weaning and is not associated with a postmenopausal decrease in BMD and osteoporosis risk. Therefore, it should be a matter of concern only for lactating women with additional risk factors or with low BMD before pregnancy.
## Conflicts of interest:
The authors declare no conflict of interest.
[fig] Figure 1: Neuroendocrine control of amenorrhea. ARC-arcuate nucleus; AVPN-anteroventral periventricular nucleus; Kiss1-kisspeptin; Kiss1r-kisspeptin receptor; GnRH-gonadotropin-releasing hormone; LH-luteinizing hormone. [/fig]
[fig] Author: Contributions: Writing-review and editing, A.C.-K., M.S., K.C., M.G., R.S.; visualization, A.C.-K., M.S., K.C., M.G., R.S.; supervision, A.C.-K., M.S., K.C., M.G., R.S. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. [/fig]
[table] Table 1: Summary of LAM acceptance and correctness in selected countries.Table 1. Cont. [/table]
[table] Table 2: Decrease in BMD depending on the site of measurements and the duration of lactation.Legend: BMD-bone marrow density; ms-months; n/d-no data; NS-no significant differences. [/table]
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A case report of primary small cell carcinoma of the breast and review of the literature
Primary small cell carcinoma (SCC) of the breast, an exceedingly rare and aggressive tumor, is often characterized by rapid progression and poor prognosis. We report a case of primary SCC of the breast that was diagnosed through pathologic and immunohistochemical examinations. Computed tomography (CT) scans failed to reveal a non鄄 mammary primary site. Due to the scant number of relevant case summaries, this type of tumor is proved to be a diagnostic and therapeutic challenge. Therefore, we also reviewed relevant literature to share expertise in diagnosis, clinicopathologic characteristics, treatment, and prognosis of this type of tumor. Future studies with more cases are required to define more appropriate treatment indications for this disease.
Primary small cell carcinoma (SCC) of the breast is a subtype of neuroendocrine carcinoma. .
[1] described the first case of SCC of the breast in 1983. To our knowledge, there are less than 60 reported cases in the literature. The diagnosis of this disease can only be made if there is no evidence for a nonmammary site or if an component can be demonstrated . Primary SCC of the breast accounts for less than 1% of primary breast cancers . Due to the rarity of this type of tumor and the lack of a standard treatment, we report here a case of primary SCC of the breast and discuss its clinicopathologic characteristics with literature review.
## Case report
In October 2011, a 39yearold woman presented a painless mass in her left breast for 3 months. There were no accompanying symptoms such as nipple retraction, dimpling, or palpable axillary lymph nodes. Physical examination revealed a 2 cm 伊 2 cm, elastic, firm, illdefined suspicious mass in the upper outer quadrant of her left breast. On the mammography, the mass appeared round with indistinct margins with lots of punctates and scattered calcifications along the duct [fig_ref] Figure 1: A, mammography reveals a round mass with obscure margins and lots of... [/fig_ref].
Additionally, an ultrasound scan revealed two solid and low heterogeneous echoes in the left breast: one was in the 3 o爷clock position, 35 mm away from the nipple [fig_ref] Figure 1: A, mammography reveals a round mass with obscure margins and lots of... [/fig_ref] ; the other was also in the 3 o爷clock position, 65 mm away from the nipple [fig_ref] Figure 1: A, mammography reveals a round mass with obscure margins and lots of... [/fig_ref]. Both were poorly defined with irregular borders. However, the levels of serum tumor markers, including carcino鄄 embryonic antigen (CEA), carbohydrate antigen 125 (CA125), CA153, and CA199 were all within normal limits.
The patient did not undergo a fine needle biopsy. An intraoperative biopsy confirmed the pathologic diagnosis of SCC with the component of ductal carcinoma (DCIS). As a result, she underwent modified radical mastectomy with axillary lymph node dissection. Microscopically, the mass (in the 3 o爷clock position, 35 mm away from the nipple) was about 0.8 cm 伊 0.5 cm 伊 0.5 cm, which was diagnosed histologically as an intermediate grade DCIS, whereas the second mass (in the 3 o爷clock position, 65 mm away from the nipple) was about 1.4 cm 伊 1.2 cm 伊 0.6 cm and was comprised Histological examination showed that the tumor was composed of small round or oval cells with a high nucleocytoplasmic ratio, small hyperchromatic nuclei, inconspicuous nucleoli, and scant cytoplasm, which were arranged diffusely and patchily. Widespread necrosis was present [fig_ref] Figure 2: A, The small cell carcinoma component [/fig_ref]. Immunohistochemically, tumor cells of the DCIS component were positive for estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). However, the tumor cells of the SCC component were negative for these markers, whereas the Ki67 proliferation index (percentage of Ki67positive cancer nuclei) was 70% . Furthermore, the SCC tumor cells were highly positive for neuroendocrine differentiation markers including synaptophysin [fig_ref] Figure 2: A, The small cell carcinoma component [/fig_ref] , neuron specific enolase (NSE) [fig_ref] Figure 2: A, The small cell carcinoma component [/fig_ref] , thyroid transcription factor1 (TTF1) [fig_ref] Figure 2: A, The small cell carcinoma component [/fig_ref] , and CD56 [fig_ref] Figure 2: A, The small cell carcinoma component [/fig_ref] , but they were negative for chromogranin A (CgA) [fig_ref] Figure 2: A, The small cell carcinoma component [/fig_ref]. Histological examination also demonstrated 1 positive lymph node out of 13 level I axillary lymph nodes. Therefore, a search for a nonmammary primary site was carried out, especially focusing on pathologic changes in the lungs. The result of computed tomography (CT) scans of the neck, chest, and upper abdomen were all within normal limits, and bone scintigraphy was negative for metastatic disease. As a result, we concluded that the breast was the primary site, and primary SCC of the breast was diagnosed.
The patient was treated with six cycles of docetaxel (75 mg/m 2 ) and carboplatin (350 mg/m 2 ) in combination once every three weeks. After the initial treatment, the patient was clinically monitored for disease relapse and metastasis.
## A
# Discussion
Small cell neuroendocrine carcinoma has been described in many extrapulmonary sites, including the breast, larynx, gastrointestinal tract, prostate, bladder, ovary, and cervix . In particular, primary SCC of the breast is considerably rare. To our knowledge, 56 cases have been reported in the literature . In summarizing the clinicopathologic characteristics of these cases, most of them are found in women; only two male cases have been reported . The median age of the patients at presentation was 52 years (range, 29 to 81 years). Tumor size ranged from 1 to 18 cm (mean, 4.63 cm), and about 62.8% of the tumors (2 to 5 cm in size) were in T2 stage according to the National Comprehensive Cancer Network (NCCN) guidelines. Approximately 60.4% (32/53) of the patients exhibited axillary lymph node metastasis. Most of them were accompanied by vascular invasion and intraductal lesion. Primary SCC of the breast can be diagnosed if there is no evidence for an extramammary primary site or if an component can be demonstrated histopathologically within the breast. It is important that the DCIS component is truly a precursor lesion of SCC and supports a primary breast origin . The presence of an component is useful, though not indispensable, toward the diagnosis of primary breast tumors.
We also reviewed previous immunohistochemical reports . The tumor cells from approximately 28.8% (15/52) of reported patients were positive for either ER or PR. However, HER2 overexpression has never been reported in these cases. In general, the positive rates of ER, PR, and HER2 were about 60% 70% , 50% 60% , and 20% 30% , respectively, displaying a common phenotype of invasive ductal carcinoma . Moreover, positive neuroendocrine markers could provide strong support for diagnosis. The positive rates of NSE and synaptophysin were 88.2% (45/51) and 63.8% (30/47), respectively, and some of the tumor cells were positive for CD56, TTF1, CgA, and other markers.
This type of breast carcinoma is rare and has generally been considered to be extremely aggressive. As a result, no standard treatment for this disease exists . The most effective option seems to be modified radical mastectomy with axillary lymph node dissection followed by adjuvant chemotherapy. Considering that the biological characteristics of this type of breast carcinoma are similar to SCC of the lung, effective drugs for SCC, such as platinum compounds and etoposide, were selected to treat these patients; drugs for common types of breast cancer were not selected. Paclitaxel has been found safe and effective as firstline therapy for small cell lung carcinoma [bib_ref] Primary small cell neuroendocrine carcinoma of the breast, Bigotti [/bib_ref]. In addition, paclitaxelcontaining regimens have also been used for patients with breast cancer as a standard adjuvant therapy. In the case of SCC with a DCIS component, we prefer a docetaxel and carboplatin combination rather than other ordinary regimens such as cyclophosphamide, epirubicin, and 5fluorouracil
(CEF). Individual treatment strategy, including chemotherapy, radiotherapy, and endocrine therapy alone or in combination, should be determined according to patient爷s age, tumor size, axillary lymph node status, and molecular phenotype.
The prognosis for this type of tumor is generally believed to be as poor as its pulmonary counterparts . However, previous reports show that the prognosis can be better if such tumors are detected at an earlier stage. According to previous reports , the mean followup time was 21 months (range, 3 to 60 months), the metastasis or relapse rate was 35.7% (20/56), and the mortality was 19.6% (11/56). The most common sites of metastasis were the liver, brain, bone, lungs, and lymph nodes. The treatment of disease relapse, however, remains poorly characterized. As for our 39yearold patient, tumor cells of the SCC component were negative for ER, PR, and HER2. In addition, the high Ki67 index and the presence of metastases in 4 axillary lymph nodes may result in a high risk of disease relapse and metastasis. Thus, after her systemic and individual treatments, a rigorous clinical monitoring of disease relapse and metastasis is necessary.
# Conclusions
In summary, primary SCC of the breast is a new challenge. Due to the rarity of cases, no standard treatment exists. According to previous reports, earlier detection and multidisciplinary therapies may be relevant to improved prognosis. Further studies with more cases will enhance our understanding of the clinicopathologic characteristics of this tumor and may result in the development of new therapeutic modalities, thus improving the outcome of these patients.
[fig] Figure 1: A, mammography reveals a round mass with obscure margins and lots of punctates and scattered calcifications along the duct. B and C, ultrasound scan shows two solid and low heterogeneous echoes in the left breast. One is in the 3 o爷clock position, 65 mm away from the nipple (B); the other is also in the 3 o爷clock position, 35 mm away from the nipple (C). Two lesions are both poorly defined with irregular borders.Qi鄄 Dong Ge et al. Small cell carcinoma of the breast of intermediate grade DCIS ( 30 % ) and SCC ( 70 % ) . [/fig]
[fig] Figure 2: A, The small cell carcinoma component (left) is comprised of small round or oval cells with hyperchromatic nuclei, scant cytoplasm, and inconspicuous nucleoli, accompanied with a ductal carcinoma in situ component (right) (HE 伊200). B-F, immunohistochemical staining of the tumor. The small cell carcinoma component (left) is positive for synaptophysin (B), neuron specific enolase (C), thyroid transcription factor鄄 1 (D), and CD56 (E) and is negative for chromogranin A (F); whereas the ductal carcinoma in situ component (right) is negative for all the above markers (IHC 伊200). [/fig]
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Multicellular Tumor Spheroids for Evaluation of Cytotoxicity and Tumor Growth Inhibitory Effects of Nanomedicines In Vitro: A Comparison of Docetaxel-Loaded Block Copolymer Micelles and Taxotere®
While 3-D tissue models have received increasing attention over the past several decades in the development of traditional anti-cancer therapies, their potential application for the evaluation of advanced drug delivery systems such as nanomedicines has been largely overlooked. In particular, new insight into drug resistance associated with the 3-D tumor microenvironment has called into question the validity of 2-D models for prediction of in vivo anti-tumor activity. In this work, a series of complementary assays was established for evaluating the in vitro efficacy of docetaxel (DTX) -loaded block copolymer micelles (BCM+DTX) and TaxotereH in 3-D multicellular tumor spheroid (MCTS) cultures. Spheroids were found to be significantly more resistant to treatment than monolayer cultures in a cell line dependent manner. Limitations in treatment efficacy were attributed to mechanisms of resistance associated with properties of the spheroid microenvironment. DTX-loaded micelles demonstrated greater therapeutic effect in both monolayer and spheroid cultures in comparison to TaxotereH. Overall, this work demonstrates the use of spheroids as a viable platform for the evaluation of nanomedicines in conditions which more closely reflect the in vivo tumor microenvironment relative to traditional monolayer cultures. By adaptation of traditional cell-based assays, spheroids have the potential to serve as intermediaries between traditional in vitro and in vivo models for high-throughput assessment of therapeutic candidates. Citation: Mikhail AS, Eetezadi S, Allen C (2013) Multicellular Tumor Spheroids for Evaluation of Cytotoxicity and Tumor Growth Inhibitory Effects of Nanomedicines In Vitro: A Comparison of Docetaxel-Loaded Block Copolymer Micelles and TaxotereH. PLoS ONE 8(4): e62630.
# Introduction
It has become increasingly clear that resistance to chemotherapy is not only facilitated by processes at the cellular level, but also by mechanisms associated with the tumor microenvironment [bib_ref] Drug resistance and the solid tumor microenvironment, Trédan [/bib_ref] [bib_ref] Tumor Microenvironment Complexity: Emerging Roles in Cancer Therapy, Swartz [/bib_ref]. In growing tumors, the heterogeneous architecture of the vasculature, irregular blood flow, large intervascular distances and nature of the extracellular matrix limit the access of cells to oxygen, nutrients, and systemically administered therapies [bib_ref] Delivering nanomedicine to solid tumors, Jain [/bib_ref] [bib_ref] Drug penetration in solid tumours, Minchinton [/bib_ref]. Within the tumor interstitium, gradients in the rate of cell proliferation are established wherein rapidly dividing cells reside close to the tumor vasculature and quiescent cells are situated deep within the extravascular space. However, many anti-neoplastic agents exert limited toxicity against slowly-or non-proliferating cells and are less effective in the hypoxic and acidic microenvironments of poorly perfused tissues [bib_ref] Hypoxia in cancer: significance and impact on clinical outcome, Vaupel [/bib_ref] [bib_ref] The role of the 3D environment in hypoxiainduced drug and apoptosis resistance, Kim [/bib_ref]. These therapeutic limitations are exacerbated by high interstitial fluid pressure which inhibits the penetration of chemotherapeutic agents through the tumor interstitium by limiting convective transport [bib_ref] Delivery of Molecular and Nanoscale Medicine to Tumors: Transport Barriers and Strategies, Chauhan [/bib_ref]. As a result cells located distant from blood vessels may be less sensitive to treatment and also be exposed to sub-therapeutic drug concentrations.
The use of in vitro cell culture is critical in drug discovery and formulation development for rapid identification of lead candidates and for investigating mechanisms of drug efficacy at the cellular and molecular levels. In contrast to in vivo tumor models, in vitro cultures are better suited for systematic studies of formulation parameters in a highly controlled environment. However, cytotoxic effects observed in conventional monolayer cultures often fail to translate into similar effects in vivo [bib_ref] Relationships between drug activity in NCI preclinical in vitro and in vivo..., Johnson [/bib_ref] [bib_ref] A Critical Appraisal of the Predictive Value of in Vitro Chemosensitivity Assays, Phillips [/bib_ref]. This is due to the inherent inability of 2-D cultures to account for mechanisms of drug resistance and transport restrictions associated with the 3-D tumor microenvironment. As such, there is increasing interest in applying 3-D in vitro models that enable rapid, high throughput screening of drug formulations for selection of lead candidates to move forward to in vivo evaluation [bib_ref] A review of three-dimensional in vitro tissue models for drug discovery and..., Elliott [/bib_ref] [bib_ref] 3D tumour models: novel in vitro approaches to cancer studies, Nyga [/bib_ref] [bib_ref] Advances in establishment and analysis of 3D tumour spheroid-based functional assays for..., Vinci [/bib_ref].
As depicted in [fig_ref] Figure 1: 3-D cultures as intermediary between 2-D cultures and animal models [/fig_ref] , 3-D tissue cultures such as MCTS serve as an intermediary between the oversimplified structure of monolayer cultures and the highly complex nature of in vivo tumors. Spheroid cultures possess a complex network of cell-cell contacts and advanced extracellular matrix development, as well as pH, oxygen, metabolic and proliferative gradients analogous to the conditions in poorly vascularized and avascular regions of solid tumors [bib_ref] Influence of hypoxia and an acidic environment on the metabolism and viability..., Rotin [/bib_ref] [bib_ref] Demonstration of an Extracellular Matrix in Multicellular Tumor Spheroids, Nederman [/bib_ref] [bib_ref] Comparative pO2 measurements in cell spheroids cultured with different techniques, Acker [/bib_ref]. In general, a spheroid is comprised of an outer region of proliferating cells which surrounds intermediate layers of quiescent cells and, if the spheroid is large enough, a necrotic core. This arrangement parallels the radial organization of tissues surrounding tumor blood vessels. To date, a variety of 3-D in vitro tissue models have been applied for the study of anticancer therapies including natural and synthetic tissue scaffolds [bib_ref] Engineering tumors with 3D scaffolds, Fischbach [/bib_ref] [bib_ref] 3-D tumor model for in vitro evaluation of anticancer drugs, Horning [/bib_ref] , multicellular layers [bib_ref] An experimental and mathematical model for the extravascular transport of a DNA..., Hicks [/bib_ref] [bib_ref] Limited penetration of anticancer drugs through tumor tissue: a potential cause of..., Tannock [/bib_ref] [bib_ref] Factors that influence the penetration of methotrexate through solid tissue, Cowan [/bib_ref] [bib_ref] Survival and structural evaluations of three-dimensional tissues fabricated by the hierarchical cell..., Chetprayoon [/bib_ref] [bib_ref] Engineering fibrotic tissue in pancreatic cancer: A novel three-dimensional model to investigate..., Hosoya [/bib_ref] , and multicellular tumor spheroids [bib_ref] Engineering tumors with 3D scaffolds, Fischbach [/bib_ref] [bib_ref] The penetration of anticancer drugs through tumor tissue as a function of..., Grantab [/bib_ref] [bib_ref] Multicellular tumor spheroids: an underestimated tool is catching up again, Hirschhaeuser [/bib_ref]. MCTS are particularly relevant in the development of nanomedicines since the penetration of the encapsulated drug in tumor tissues may be significantly altered by properties of the delivery vehicle. To date, however, there remain limited examples of the use of MCTS for the evaluation of nanomedicines [bib_ref] The delivery of doxorubicin to 3-D multicellular spheroids and tumors in a..., Kim [/bib_ref] [bib_ref] Accumulation and toxicity of antibodytargeted doxorubicin-loaded PEG-PE micelles in ovarian cancer cell..., Perche [/bib_ref] [bib_ref] Enhanced growth inhibition of hepatic multicellular tumor spheroids by lactosylated poly(ethylene glycol)-siRNA..., Oishi [/bib_ref] [bib_ref] Pharmaceutical and biomedical differences between micellar doxorubicin (NK911) and liposomal doxorubicin (Doxil), Tsukioka [/bib_ref] [bib_ref] Preparation and biological characterization of polymeric micelle drug carriers with intracellular pH-triggered..., Bae [/bib_ref].
DTX is a potent chemotherapeutic agent that is administered as TaxotereH (Sanofi-Aventis) and used for treatment of cancers of the breast, prostate, lung, head and neck, and stomach. DTX is also being investigated in a phase II clinical trial for treatment of metastatic colorectal adenocarcinoma in combination with gemcitabine and has been investigated as a single agent for treatment of cervical cancer. However, TaxotereH is known to be associated with significant side effects that can require reduction of the administered dose [bib_ref] A phase II trial of docetaxel (Taxotere) as second-line chemotherapy in patients..., Baur [/bib_ref]. Encapsulation of chemotherapeutic agents within biocompatible nanosystems such as block copolymer micelles (BCMs) has proven to be a promising approach for mitigating the burden of toxicity on normal tissues and increasing tumor-specific drug accumulation [bib_ref] Block copolymer micelles for delivery of cancer therapy: transport at the whole..., Mikhail [/bib_ref]. The primary objective of this study was to adapt and apply traditional cell-based assays in a systematic and complementary manner for the evaluation of TaxotereH and a DTX-containing nanomedicine in both monolayer and MCTS cultures [fig_ref] Figure 2: In vitro assays used in this study for analysis of formulation efficacy... [/fig_ref].
# Materials and methods
# Materials
Methoxy poly(ethylene glycol) (CH 3 O-PEG-OH; Mn = 5000, Mw/Mn = 1.06) was obtained from Sigma-Aldrich (Oakville, ON, Canada). e-Caprolactone and dichloromethane (Sigma-Aldrich) were dried using calcium hydride prior to use. Hydrogen chloride (HCl) (1.0 M in diethyl ether), N,N-dimethylformamide (DMF), diethyl ether, hexane and acetonitrile (Sigma-Aldrich) were used without further purification. Alexa Fluor 488 (AF488) carboxylic acid succinimidyl ester was purchased from Molecular Probes (Eugene, OR). The hypoxia marker, EF5, and Cy5-conjugated anti-EF5 antibody were purchased from the Department of Radiation Oncology, University of Pennsylvania, (Philadelphia, PA). DTX was purchased from Jari Pharmaceutical Co. (Jiangsu, China).
## Synthesis of ch 3 o-peg-b-pcl (peg-b-pcl) copolymers
PEG-b-PCL copolymer was prepared as previously described [bib_ref] Poly(ethylene glycol)-b-poly(epsilon-caprolactone) micelles containing chemically conjugated and physically entrapped docetaxel: synthesis, characterization,..., Mikhail [/bib_ref]. Briefly, CH 3 O-PEG-OH was used to initiate the ringopening polymerization of e-CL in the presence of HCl. The reaction was carried out for 24 h at room temperature prior to termination by addition of triethylamine (TEA) and precipitation in diethyl ether and hexane (50:50, v/v%). The product was dried under vacuum at room temperature.
## Preparation and characterization of bcm+dtx
PEG-b-PCL copolymers and DTX were dissolved at a copolymer:drug weight ratio of 20:1 in DMF and stirred for 30 min. DMF was evaporated under N 2 at 30uC and residual solvent was removed under vacuum. Dry copolymer-drug films were then heated to 60uC in a water bath prior to the addition of PBS buffer (pH 7.4) at the same temperature. Resultant micelle solutions were vortexed, stirred for 24 h at room temperature and finally sonicated (Laboratory Supplies Co., NY) for 1 h. Undissolved drug crystals were removed by centrifugation at 4400 g for 12 min (Eppendorf 5804R). The final copolymer concentration was 10 mg/mL. The amount of physically entrapped DTX in BCM samples was determined by HPLC analysis (Agilent series 1200) with UV detection (Waters 2487) at a wavelength of 227 nm. An XTerra C18 reverse phase column was employed with ACN/ water (60/40, v/v%) as the mobile phase. Drug loading was quantified using a calibration curve generated from a series of DTX standards.
## Sizing of bcm+dtx
The average hydrodynamic diameter of the BCMs was determined by dynamic light scattering (DLS) using a 90Plus Particle Size Analyzer (Brookhaven Instruments Corp., Holtsville, NY) at an angle of 90u and temperature of 37uC. The samples were diluted to a copolymer concentration of 0.5 mg/mL prior to measurement. Analysis was performed using the 90Plus Particle Sizing Software.
## Transmission electron microscopy (tem)
BCMs were observed by TEM using a Hitachi 7000 microscope operating at an acceleration voltage of 75 kV (Schaumburg, IL). Samples were diluted in double distilled water immediately prior to analysis and negatively stained with a 1% uranyl acetate (UA) solution. The final copolymer concentration was 0.5 mg/mL. The samples were then deposited on copper grids that had been pre-
## Drug release
The release of DTX from BCMs and TaxotereH was analyzed using a dialysis method. Aliquots (1 mL) of BCM+DTX, DTX in DMSO, and TaxotereH were placed in individual dialysis bags (MWCO 2 kDa, Spectra/Por, Rancho Dominguez, CA) and dialyzed separately against 100 mL of PBS at pH 7.4 in an incubator at 37uC ensuring that sink conditions were maintained. At selected timepoints, 50 mL samples were withdrawn from the dialysis bags and DTX content was measured by HPLC as described above.
## Tissue culture and growth of mcts
Human cervical (HeLa) and colon (HT29) (ATCC, Manassas, VA) cancer cells were incubated at 37uC and 5% CO 2 in DMEM containing 1% penicillin-streptomycin and supplemented with 10% FBS. For growth of MCTS, cells were suspended using trypsin-EDTA and 2000 and 5000 HT29 and HeLa cells were seeded onto non-adherent 96-well round-bottomed Sumilon PrimeSurface TM plates (Sumitomo Bakelite, Tokyo, Japan), respectively, in 200 mL of media per well. During growth, 50% of the media was exchanged every other day. MCTS were grown for 7 days until they reached , 500 mm in diameter before use.
## Immunohistochemical analysis of mcts
MCTS were washed in PBS and transferred onto a vinyl specimen mold (CryomoldH, Tissue-Tek, Sakura Finetek, CA) prior to addition of Tissue-TekH O.C.T. compound (Sakura Finetek, Torrance, CA). MCTS were then submersed in an isopentane bath cooled by liquid nitrogen, cut into 5 mm thick sections using a microtome and mounted on glass slides. Histological staining was conducted for the identification of cellular proliferation (Ki67) and stained with hematoxylin and eosin (H&E). For identification of hypoxic regions, MCTS were incubated with 0.5 mM EF5 and soaked in PBS prior to cryosectioning. EF5 in the MCTS sections was identified by binding with cyanine-5-conjugated mouse anti-EF5 (1/50) antibody. The positive signal distribution for Ki67 was analyzed using a customized MATLABH algorithm, as described previously [bib_ref] An injectable depot system for sustained intraperitoneal chemotherapy of ovarian cancer results..., Zahedi [/bib_ref]. Briefly, images containing Ki67-stained MCTS sections were thresholded for positive color intensity. Using a distance map, signal intensities were summed within three concentric regions of equidistant thickness (periphery, intermediate and core), each equivalent to 1/3 of the MCTS radius. The distribution of Ki67 positive signal is expressed as a percentage of total positive signal in the MCTS section.
## Measurement of mcts growth
Spheroids were imaged using a light microscope with a 106 objective lens (VWR VistaVision TM) connected to a digital camera (VWR DV-2B). The diameter and volume of MCTS were determined by measuring their cross-sectional area using an automated image analysis macro developed for use with the ImageJ software package (NIH, Bethesda, MD, Version 1.44 m). The automated method was validated by comparison to manual determination of spheroid diameter and volume [fig_ref] Figure 1: 3-D cultures as intermediary between 2-D cultures and animal models [/fig_ref]. For the automated method, images were converted into 8-bit greyscale and the perimeter of an individual MCTS was recognized by an automated threshold function and the image converted to a 2-D mask. The area of the spheroid mask was recorded, applying an image of known scale as calibration. Finally, the volume of the MCTS was calculated by assuming a spherical shape as follows: V = 4/3*p*(d/2) 3 . Data was fit using the Gompertz equation for tumor growth as follows: V(t) = V(0)exp(a/b(12exp(2b*t))) where V(t) is volume at time t, V(0) the initial volume and a and b are constants [bib_ref] Analysis of growth of multicellular tumour spheroids by mathematical models, Marusić [/bib_ref].
## Cytotoxicity in monolayer and spheroid tissue cultures
The cytotoxicity of BCM+DTX and TaxotereH in monolayer and spheroid cell cultures was determined using the established acid phosphatase (APH) assay which is based on quantification of cytosolic acid phosphatase activity [bib_ref] A reliable tool to determine cell viability in complex 3-d culture: the..., Friedrich [/bib_ref]. For this assay, pnitrophenyl phosphate is added in cell culture and hydrolyzed in viable cells to p-nitrophenol via intracellular acid phosphatase. Briefly, MCTS (one spheroid per well) and monolayer cultures (4000 cells per well) were treated with TaxotereH or BCM+DTX for 24 h over a range of drug concentrations. Following treatment, monolayers and spheroids were washed three times with fresh media and cultured for an additional 48 h. Monolayers and spheroids were then washed with PBS buffer prior to the addition of 100 mL of freshly prepared reaction buffer (2 mg/ml pnitrophenyl phosphate (Sigma) and 0.1% v/v Triton-X-100 in 0.1 M sodium acetate buffer at pH 5.5). Following incubation for 2 h in the cell incubator, 10 mL 1 M sodium hydroxide was added to each well and cell viability was determined by measuring the UV absorbance at 405 nm using an automated 96-well plate reader (SpectraMax Plus 384, Molecular Devices, Sunnyvale, CA). Results were normalized to controls as follows: % viability = (Atreatment -A media )/(A control -A media ), where A = mean absorbance. All experiments were performed in triplicate.
## Growth inhibition of mcts
BCM+DTX or TaxotereH was administered to spheroids for 24 h at a DTX equivalent concentration of 2, 20 or 200 ng/ mL. The culture media was replaced following the incubation period. Subsequently, half of the culture media was replaced by pipette every other day. Images of spheroids were captured using a light microscope with a 106 objective lens (VWR VistaVision TM ) connected to a digital camera (VWR DV-2B). Spheroid size was determined by measuring their 2-D crosssectional area using the automated image analysis method described previously. The data are reported as the mean volume of six spheroids 6 SD.
## Clonogenic survival assay
The clonogenic assay was used to determine the ability of single cells to replicate and form colonies (.50 cells) following exposure to BCM+DTX and TaxotereH. Single cell suspensions derived from monolayer and disaggregated spheroids were diluted in culture media and cells were plated in 6-well plates in desired numbers. MCTS were disaggregated by incubation in trypsin-EDTA for 10 min, followed by gentle agitation. Drug formulations were added immediately at a DTX equivalent concentration of 20 ng/mL. After treatment for 24 h, cells were washed with PBS and 2 mL of fresh media was added to each well. For treatment of intact spheroids, drug formulations were added directly into wells containing individual MCTS. After 24 h, MCTS were collected and rinsed in PBS, suspended as single-cell suspensions in fresh media following trypsinization, and seeded onto 6-well plates. Cells were incubated for 14-16 days prior to fixation with methanol and staining with 1% crystal violet solution. Colonies consisting of at least 50 cells were counted. The surviving fraction (SF) was expressed as the number of colonies divided by the product of the number of cells plated and the plating efficiency. The plating efficiency was determined by dividing the number of colonies formed by the number of cells plated for untreated controls.
# Results
## Characterization of bcm+dtx
PEG-b-PCL copolymer micelles containing physically encapsulated DTX were formulated with a spherical morphology [fig_ref] Figure 3: Characterization of micelle morphology and size [/fig_ref]. The size distribution of the micelles was monomodal with an average hydrodynamic diameter of 49.262.3 nm [fig_ref] Figure 3: Characterization of micelle morphology and size [/fig_ref]. Drug loading resulted in a final DTX equivalent concentration of 258.7635.5 mg/mL at a loading efficiency of 52.767.1%. Release of DTX from BCMs occurred over the course of 24 h wherein 74% of the drug was released by 12 h. In contrast, the release of docetaxel from TaxotereH was complete by 12 h [fig_ref] Figure 4: Drug release [/fig_ref].
## Growth of mcts
Spheroids were grown using a modified liquid overlay technique by seeding HT29 or HeLa cells onto non-adherent U-bottom tissue culture wells without the use of an agarose surface coating. MCTS were spherical, followed a sigmoidal growth profile, and were grown until a diameter of ,500 mm was reached prior to use [fig_ref] Figure 5: Spheroid packing density and growth [/fig_ref].
## Cytotoxicity in monolayer and mcts culture
Cell viability following exposure to BCM+DTX or TaxotereH was assessed using the APH assay [fig_ref] Figure 6: Cytotoxicity of TaxotereH and BCM+DTX in spheroid and monolayer cultures [/fig_ref]. This assay was validated by assessment of the relationship between UV absorbance and cell number in both monolayer and spheroid cultures. As shown in [fig_ref] Figure 2: In vitro assays used in this study for analysis of formulation efficacy... [/fig_ref] , a linear relationship was obtained. A wellestablished tetrazolium salt-based assay (WST-8) was also evaluated and did not yield a similar correlation [fig_ref] Figure 3: Characterization of micelle morphology and size [/fig_ref]. Spheroid cultures were substantially less sensitive to BCM+DTX and TaxotereH relative to their monolayer counterparts. HeLa cells were less responsive to treatment with either BCM+DTX or TaxotereH than HT29 cells in monolayer culture. However, in spheroid culture, HT29 cells were less sensitive to treatment. The IC [bib_ref] Entrapment of B-lactams antibiotics in polyethylcyanoacrylate nanoparticles: Studies on the possible in..., Cavallaro [/bib_ref]
## Inhibition of mcts growth
MCTS volume was plotted over a 30 day period following a 24 h incubation with 2, 20, and 200 ng/mL BCM+DTX or TaxotereH [fig_ref] Figure 7: Inhibition of spheroid growth [/fig_ref]. The growth of HeLa MCTS was completely impeded following incubation with DTX concentrations of 20 and 200 ng/mL. No significant difference in growth was observed following exposure to 2 ng/mL of DTX relative to untreated controls. In the case of HT29 MCTS, incubation with 20 ng/mL of BCM+DTX and TaxotereH only resulted in a partial reduction in MCTS volume. Similarly to HeLa MCTS, complete inhibition of growth was observed following incubation with 200 ng/mL of drug. Unlike HeLa MCTS, however, a slight growth delay was also observed at 2 ng/mL. Interestingly, following re-treatment on day 14 at a DTX concentration of 20 ng/mL, BCM+DTX demonstrated greater inhibition of spheroid growth in HT29 cultures than TaxotereH.
## Immunohistochemistry
Immunohistochemical analysis of MCTS cross-sections was performed in order to identify regions of necrosis, cellular proliferation and hypoxia [fig_ref] Figure 8: Histological assessment of spheroid microenvironment [/fig_ref]. Staining with the proliferation marker Ki67 revealed a greater proportion of proliferative cells in HeLa MCTS relative to HT29. Quantitative image analysis revealed that 88.6% of proliferating cells were located within the periphery of HT29 MCTS [fig_ref] Figure 9: Spatial distribution of proliferating cells in spheroids [/fig_ref]. In contrast, only 51% of the total proliferating cells were located in the periphery of HeLa MCTS and 25% and 24% were located in the intermediate region and core, respectively. Signs of necrosis were visible following staining with H&E in HT29 MCTS. Incubation of MCTS with EF5 allowed for identification of regions of hypoxia following exposure to Cy5-conjugated anti-EF5 antibody. Hypoxic conditions were observed primarily in the core and intermediate regions of HT29 MCTS. In contrast, HeLa MCTS did not demonstrate any regional hypoxia. The relative distributions of cellular proliferation, hypoxia and necrosis in the MCTS are summarized in [fig_ref] Figure 1: 3-D cultures as intermediary between 2-D cultures and animal models [/fig_ref].
## Clonogenic survival
The surviving fractions (SF) of HeLa and HT29 cells were determined following treatment with BCM+DTX or TaxotereH as monolayer and MCTS cultures [fig_ref] Figure 1: 3-D cultures as intermediary between 2-D cultures and animal models [/fig_ref].
The SF was higher for all intact MCTS cultures relative to monolayers. HeLa cells were less sensitive to treatment than HT29 when cultured as monolayers, but more sensitive than HT29 cells when the cells were exposed to treatment as MCTS. In all cases, the SF was lower when treated with BCM+DTX compared to TaxotereH. Furthermore, cells exposed to treatment immediately following MCTS disaggregation demonstrated residual resistance to both BCM+DTX and TaxotereH.
# Discussion
In recent years, the tumor microenvironment has been implicated in the coordination of tumor growth, metastasis and resistance to anti-cancer therapies [bib_ref] Microenvironmental regulation of metastasis, Joyce J A [/bib_ref] [bib_ref] Hallmarks of cancer: the next generation, Hanahan [/bib_ref]. As such, effective evaluation of novel therapeutic agents requires the use of tissue models which closely mimic native conditions within the intratumoral space. Yet, the vast majority of chemotherapeutic agents are screened for cytotoxic effects in monolayer cultures which do not account for critical mechanisms of drug resistance associated with the tumor microenvironment. Consequently, these models poorly predict a drug's therapeutic efficacy in vivo [bib_ref] Relationships between drug activity in NCI preclinical in vitro and in vivo..., Johnson [/bib_ref]. In contrast, 3-D MCTS better approximate the state of cancer cells in their native environment and thus can be used to more accurately estimate a drug's therapeutic potential. A variety of methods have been used to grow MCTS for use in cancer research including spinning culture flasks [bib_ref] A novel method for faster formation of rat liver cell spheroids, Okubo [/bib_ref] , hanging drops [bib_ref] Spheroid preparation from hanging drops: characterization of a model of brain tumor..., Duca [/bib_ref] , liquid overlay on agarose [bib_ref] Spheroid-based drug screen: considerations and practical approach, Friedrich [/bib_ref] , micropatterned plates [bib_ref] Microarrays for the scalable production of metabolically relevant tumour spheroids: a tool..., Hardelauf [/bib_ref] , and recently, using intercellular linkers [bib_ref] Engineering a scaffold-free 3D tumor model for in vitro drug penetration studies, Ong [/bib_ref]. However, many of these techniques are impractical, time-consuming, and involve delicate handling procedures, limiting the use of the MCTS model in drug screening and development. In addition, practical application of traditional cell-based assays in MCTS cultures remains poorly established. In the current study, the performance of BCM+DTX and TaxotereH was evaluated by adaptation of conventional cytotoxicity and survival assays in monolayer and MCTS cultures using a robust MCTS culture technique.
MCTS grew according to sigmoidal growth patterns reflective of tumor growth in vivo [fig_ref] Figure 5: Spheroid packing density and growth [/fig_ref] and possessed histological features similar to those of the native tumor microenvironment including gradients in cell proliferation and regions of hypoxia and necrosis [fig_ref] Figure 8: Histological assessment of spheroid microenvironment [/fig_ref] , [fig_ref] Figure 4: Drug release [/fig_ref]. Cells grown in spheroid cultures demonstrated considerably greater resistance to treatment with BCM+DTX or TaxotereH relative to cells grown in monolayer cultures. This may be a result of the limited exposure of cells within MCTS to treatment due to poor penetration of DTX or BCMs, the limited sensitivity of cells within MCTS to DTX due to a reduction in cellular proliferation and/or resistance associated with 3-D cell adhesion (i.e. contact effect). In a study by Kyle et al., the penetration half-depth (the depth from the surface at which the amount of drug falls to half of its maximum concentration) of DTX in multicellular layers was found to be ,25 mm following a 2 h incubation at a concentration of 0.3 mM [bib_ref] Limited tissue penetration of taxanes: a mechanism for resistance in solid tumors, Kyle [/bib_ref]. Peak tissue levels did not increase proportionally following a 10-fold increase in drug concentration although the depth of penetration was improved indicating partial saturation of tissue binding. Therefore, it is likely that high intracellular binding and consumption of DTX by peripheral cells in the MCTS limits the toxicity to cells distant from the surface. For drugs which are rapidly consumed by cells, encapsulation in BCMs which minimize interactions and uptake by cells may improve drug penetration [bib_ref] Enhanced percolation and gene expression in tumor hypoxia by PEGylated polyplex micelles, Han [/bib_ref]. For example, Pun et al. reported ameliorated penetration of doxorubicin into MCTS when encapsulated in triblock copolymer micelles [bib_ref] The delivery of doxorubicin to 3-D multicellular spheroids and tumors in a..., Kim [/bib_ref]. However, BCMs which pene- trate poorly through tissues may limit the penetration of the encapsulated drug. Overall, the extent to which the BCMs influence drug penetration will depend on the relative rates of drug release and BCM penetration in the MCTS. We have previously found that PEG-b-PCL BCMs of 55 nm diameter can achieve a homogeneous distribution in MCTS following a 24 h incubation (unpublished data).
In addition to potential limitations in MCTS penetration associated with the drug and BCMs, the discrepancy between MCTS and monolayer cytotoxicity may also be a result of drug resistance imparted by the MCTS microenvironment. A marked decrease in the proportion of proliferating cells was observed in MCTS with increasing depth from the surface [fig_ref] Figure 9: Spatial distribution of proliferating cells in spheroids [/fig_ref]. Since DTX exerts its therapeutic effect on cycling cells, cells located near the MCTS surface will respond to treatment similarly to cells cultured as monolayers. By contrast, quiescent cells that are located in the intermediate and core regions of the MCTS will be less sensitive to treatment. This notion is supported by the observation that cells exposed to treatment immediately following disaggregation of MCTS demonstrated greater clonogenic survival than monolayer cells, but less than cells treated as intact MCTS. Therefore, there exists a population of cells within the MCTS that is more resistant to treatment than cells cultured as monolayers even in the absence of any physical barrier to drug penetration. As such, the limited sensitivity of MCTS to treatment is likely a result of both restricted transport and mechanisms of drug resistance associated with the MCTS microenvironment.
The extent to which culturing cells as MCTS influenced the therapeutic effect of BCM+DTX and TaxotereH relative to monolayers was found to be cell-line specific. In monolayer cultures, BCM+DTX and TaxotereH demonstrated greater cytotoxicity against HT29 cells relative to HeLa cells. In contrast, culturing cells as MCTS imparted a greater enhancement in therapeutic resistance (i.e. greater increase in IC 50 ) to HT29 cells than to HeLa cells. We have previously shown significantly greater penetration of BCMs into HeLa MCTS than HT29 MCTS due to the former's lower cell packing density and large intercellular channels (unpublished data). In the current study, significant cell line-dependent differences in MCTS microenvironment were observed. Limited permeability of HT29 MCTS and/or high consumption of oxygen by peripheral cells was reflected by the presence of central hypoxia and necrosis. Importantly, HT29 MCTS contained a greater proportion of non-proliferating cells relative to HeLa MCTS. It is likely that some quiescent cells within the MCTS retained their clonogenic potential following exposure to sub-therapeutic amounts of DTX and were capable of recommencing proliferative activity when re-plated as monolayers. The greater clonogenic potential of HeLa cells following disaggregation of MCTS relative to HT29 cells likely reflects the greater sensitivity of HT29 monolayer cells to DTX rather than greater residual resistance of MCTS-derived HeLa cells.
One of the important advantages of the MCTS model is that it allows for treatment efficacy to be observed over an extended period of time. In order to evaluate the potential of surviving cells to repopulate MCTS, the growth of MCTS following treatment with BCM+DTX and TaxotereH was evaluated for 28 days with treatment re-applied after 14 days. The results of this study demonstrate both dose-and time-dependent changes in MCTS growth following incubation with the drug formulations. Near complete elimination of HeLa MCTS was observed following treatment at 20 ng/mL or greater with either BCM+DTX or TaxotereH. In contrast, only partial growth inhibition was observed in HT29 MCTS when exposed to the same concentration. This observation is consistent with the results obtained from the cytotoxicity and clonogenic assays in which HT29 MCTS demonstrated greater resistance to treatment relative to HeLa MCTS. A slight inhibitory effect in HT29 MCTS following administration of DTX formulations at 2 ng/mL was likely due to the cytotoxicity and shedding of surface cells, consistent with the response of HT29 cells to treatment in monolayer cultures. In addition, the apparent discrepancy between the limited cytotoxicity in HT29 spheroids revealed using the APH assay (measured 2 days post drug incubation) and the marked growth inhibition at 20 ng/mL is consistent with the observed 4 day delay in growth inhibitory effect. Interestingly, little difference in spheroid growth inhibition was observed between BCM+DTX and TaxotereH following initial treatment. It should be noted, however, that following retreatment after 14 days of culture, BCM+DTX demonstrated a greater growth inhibitory effect relative to TaxotereH.
Several factors may have contributed to the greater cytotoxicity of BCM+DTX relative to TaxotereH in monolayer and MCTS cultures. It has been hypothesized that DTX is taken up more rapidly by cells following release from BCMs in close proximity to the cell membrane due to an increase in the local transmembrane concentration gradient [bib_ref] Paclitaxel-loaded PLGA nanoparticles: preparation, physicochemical characterization and in vitro anti-tumoral activity, Fonseca [/bib_ref] [bib_ref] Reversion of multidrug resistance using nanoparticles in vitro: influence of the nature..., Nemati [/bib_ref] [bib_ref] Entrapment of B-lactams antibiotics in polyethylcyanoacrylate nanoparticles: Studies on the possible in..., Cavallaro [/bib_ref]. Slower efflux of BCM-encapsulated DTX relative to free DTX, by avoidance of membrane efflux pumps, may also contribute to the greater therapeutic effect of the DTX-loaded BCMs [bib_ref] Dynamics of endocytosis and exocytosis of poly(D,L-lactide-co-glycolide) nanoparticles in vascular smooth muscle..., Panyam [/bib_ref] [bib_ref] Folate-receptor-targeted delivery of doxorubicin nanoaggregates stabilized by doxorubicin-PEG-folate conjugate, Yoo [/bib_ref] [bib_ref] Folate receptor targeted biodegradable polymeric doxorubicin micelles, Yoo [/bib_ref]. While these results are promising, further investigation is required to fully elucidate the mechanism of cytotoxicity that lead to enhanced therapeutic effects of BCM+DTX relative to TaxotereH in vitro.
Overall, as outlined in [fig_ref] Figure 2: In vitro assays used in this study for analysis of formulation efficacy... [/fig_ref] , each of the three assays employed in this study is unique and together they provide complementary information on the therapeutic potential of drug formulations. Importantly, comparison of results obtained in monolayer and spheroid cultures demonstrated the important influence of the microenvironment and 3-D tissue structure on formulation efficacy. Therefore, 3-D cultures such as MCTS may serve as important tools for investigating the performance of nanomedicines in environments that more closely mimic intratumoral conditions in vivo. However, while spheroids share several important structural and microenvironmental properties with native tumors, there are important differences which may limit the extent to which this in vitro model can be used to predict drug efficacy in vivo. Notably, the MCTS model does not account for the potential influence of convective flow or presence of stromal cells on drug and nanoparticle transport. Despite these limitations, evaluation of formulation efficacy in spheroids rather than monolayer cultures is expected to more accurately reflect therapeutic performance in vivo. [fig_ref] Figure 1: 3-D cultures as intermediary between 2-D cultures and animal models [/fig_ref] Measurement of spheroid volume. a) Schematic representation of the analysis process using a macro developed for ImageJ (version 1.44 m). b) Correlation between manual and automated volume measurements of HeLa MCTS. MCTS were imaged at selected intervals of growth. Manual measurement of MCTS volume was performed by determining the average of the largest and smallest diameters using the captured images and assuming a spherical MCTS morphology. Automated volume measurement was achieved using an image recognition technique in ImageJ. Firstly, MCTS images were converted into 8-bit greyscale and the perimeter of the MCTS was recognized by an automated threshold function. The area of the 2-D MCTS mask was recorded and converted to mm 2 by calibration using an image of known scale and subsequently used to calculate the volume. (TIF) [fig_ref] Figure 2: In vitro assays used in this study for analysis of formulation efficacy... [/fig_ref] Validation of the acid phosphatase (APH) assay. Results from the APH assay using HeLa (left column) and HT29 cells (right column) grown as spheroids (top row) and monolayers (bottom row) demonstrate a linear relationship between cell number and UV absorption at 405 nm. Each data point represents the mean of three independent experiments 6 SD (n = 3). (TIF) [fig_ref] Figure 3: Characterization of micelle morphology and size [/fig_ref] Failure of WST-8 assay. Results from the WST-8 assay demonstrate a non-linear correlation between the number of cells and OD 450 in spheroid culture. (TIF) [fig_ref] Figure 4: Drug release [/fig_ref] Fluorescence images of HT29 (a) and HeLa (b) tumor xenografts displaying markers of hypoxia (EF5 -blue) and blood vessels (CD31 -red). Scale bars represent 100 mm.
## Supporting information
## (tif)
Author Contributions
[fig] Figure 1: 3-D cultures as intermediary between 2-D cultures and animal models. Intermediate in complexity, 3-D cultures permit the systematic, high-throughput assessment of formulation properties in a controlled environment that approximates important properties of in vivo tumors in the absence of complex parameters which may confound data interpretation. doi:10.1371/journal.pone.0062630.g001coated with carbon and negatively charged (Ted Pella Inc., Redding, CA) and briefly air-dried prior to analysis. [/fig]
[fig] Figure 2: In vitro assays used in this study for analysis of formulation efficacy in spheroids. doi:10.1371/journal.pone.0062630.g002 [/fig]
[fig] Figure 3: Characterization of micelle morphology and size. a) Transmission electron micrograph (Scale bar in represents 100 nm) and b) size distribution of BCM+DTX as determined by dynamic light scattering at 37uC. doi:10.1371/journal.pone.0062630.g003 [/fig]
[fig] Figure 4: Drug release. Release of docetaxel from dialysis bags containing BCM+DTX, TaxotereH, and DTX in DMSO, n = 3. doi:10.1371/journal.pone.0062630.g004 [/fig]
[fig] Figure 5: Spheroid packing density and growth. a) Cells per HeLa and HT29 spheroid of given volume, n = 12. b) Growth of HeLa and HT29 spheroids, n = 6. Data was fit using the Gompertz equation for tumor growth. The dashed lines indicate spheroid properties used in the studies. doi:10.1371/journal.pone.0062630.g005 [/fig]
[fig] Figure 6: Cytotoxicity of TaxotereH and BCM+DTX in spheroid and monolayer cultures. Viability of a) HeLa and b) HT29 cells cultured as monolayers and spheroids as measured using the APH assay. Data is expressed as the percent viability relative to untreated controls and fit to the Hill equation. c) Cytotoxicity of blank PEG-b-PCL micelles as a function of copolymer concentration. Each plot represents the mean of three independent experiments 6 SD (n = 3). doi:10.1371/journal.pone.0062630.g006 [/fig]
[fig] Figure 7: Inhibition of spheroid growth. a) Sequential images of the same HeLa and HT29 spheroids following treatment with BCM+DTX at a concentration of 20 ng/mL. Bars represent 100 mm. Growth inhibition of HeLa (b,c) and HT29 (d,e) MCTS by BCM+DTX and TaxotereH at concentrations of 2, 20 and 200 ng/mL. Cells were re-treated after two weeks (arrow). Box represents expanded region of plots b) and d). Data is expressed as the mean volume of six spheroids (n = 6) 6 SD. ''*'' represents a significant difference between BCM 20 and TAX 20, p,0.05. doi:10.1371/journal.pone.0062630.g007 [/fig]
[fig] Figure 8: Histological assessment of spheroid microenvironment. HeLa (a-c) and HT29 (d-f) MCTS cross-sections stained with H&E (a, d), Ki67 proliferation marker (b, e) and EF5 (c, f), a marker of hypoxia. Scale bars represent 100 mm. g) Properties of the spheroid microenvironment and their spatial distribution. ''++'', ''+'', and ''-'', indicate high, intermediate and low levels of the corresponding feature, respectively. doi:10.1371/journal.pone.0062630.g008 [/fig]
[fig] Figure 9: Spatial distribution of proliferating cells in spheroids. Ki67 positive signal distribution relative to radial position in a) HeLa and b) HT29 MCTS as a percent of total positive stain, n = 6. doi:10.1371/journal.pone.0062630.g009 [/fig]
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Plain packaging of tobacco products: the logical next step for tobacco control policy in India
# Abstract
India implemented larger 85% pictorial health warnings on all tobacco products from 1 April 2016. However, to remove the last bit of glamour and attraction from the tobacco packs, it must now embrace plain packaging. Plain packaging prevents tobacco packs from carrying the tobacco industry brand imagery as mobile billboards. Postimplementation of larger 85% pictorial health warnings on all tobacco products, this analysis was undertaken to assess the feasibility of plain packaging as the next logical tobacco control policy measure in India. As part of this analysis, the research team reviewed the available literature on legal and policy challenges to plain packaging as a tobacco control policy initiative for India. Literature from 2010 to 2016 in English language was reviewed, which reveals that, India has taken several preparatory steps implemented by other countries like Australia and the UK that have introduced plain packaging, for example, stronger smoke-free laws, ban on tobacco advertising, promotion and sponsorship, increase in taxes and a report from civil society task force on plain packaging. The trade and investment agreements signed by India are also within the international trade norms relating to public health. A Private Member's Bill on plain packaging is also pending in the Parliament of India. Other potential challenges against such policy decision, for example, freedom of trade, right to property, violation of competition law and other laws including consumer protection laws, were found unsubstantiated by the research team. Plain packaging is the next logical step for tobacco control policy in India.
# Introduction
Tobacco is the only legal product that kills over a million people and impoverishes around 15 million people annually in India. [bib_ref] Counting 15 million more poor in India, thanks to tobacco, John [/bib_ref] The total economic costs attributable to tobacco use from all diseases in India in 2011 were estimated at US$22.4 billion.Yet, India remains one of the largest consumers of tobacco products in the world, second only to China. It is also one of the major growers of tobacco in the world.
Tobacco smoking is the second leading cause of preventable deaths globally.In response to this global epidemic, WHO developed and adopted the Framework Convention on Tobacco Control (FCTC) in 2003. The WHO FCTC outlines proven, cost-effective and evidence-based measures to combat this epidemic.From among the various evidence-based tobacco control measures recommended under the WHO FCTC, there has been an increasing interest among countries, including India, to implement standardised packaging or plain packaging to fight against the growing tobacco epidemic.As the term indicates, plain packaging aims to remove the last bit of glamour and attraction from all the tobacco packaging and prevent tobacco packs from carrying the tobacco industry brand imagery as mobile billboards.This policy analysis was undertaken to assess the feasibility of introducing plain packaging in India and to assess if this public health measure contravenes any existing Indian legislation. This policy analysis also envisions identifying the key barriers and options to address them-in the backdrop of the Australian experience, and the global interest and summary box ► Australia, the UK and several other countries have either implemented or plan to implement plain packaging of tobacco products. ► India has taken several preparatory tobacco control steps, which indicate its readiness to implement plain packaging of tobacco products. ► Existing domestic legislation and international trade obligations of India are not in conflict with plain packaging policy. ► Experience from other jurisdictions indicates tobacco industry as the major roadblock to implementing plain packaging. ► This paper will help governments and tobacco control advocates in low-income and middle-income countries, in working towards implementation of plain packaging of tobacco products.
BMJ Global Health emphasis on plain packaging of tobacco products. The study team identified and reviewed the national laws on plain packaging in Australia,the UKThe national and international laws and policies that could potentially be used by the tobacco industry to challenge a decision of implementing plain packaging in India were also identified and reviewed. Information was collected based on the recommendations of the Australia-India Institute Taskforce 13 on Tobacco Control and an iterative method of web-based searching. Copies of the written policies regulating plain packaging in Australia were reviewed by the study team to understand how plain packaging has been introduced and regulated there.The study team reviewed legislation and regulations, which were likely to be used by the Indian tobacco industry to oppose plain packaging.
## Plain packaging in australia
Canada was the first country to moot the idea of plain packaging of tobacco products, [bib_ref] The case for plain packaging, Cunningham [/bib_ref] but it was Australia which became the first country to implement the provision from 1 December 2012. Despite having little basis for legal challenge, the tobacco industry vehemently opposed the plain packaging initiative in Australia on the ground of being in contravention of constitutional rights, the Trade-Related Intellectual Property Rights (TRIPS) Agreement and the Paris Convention, leading to substantial claims for compensation against the Australian Government. However, the Australian Government, based on recommendations of the National Preventative Health Taskforce, mandated standard plain packaging of all tobacco products to inter alia, reduce the appeal of tobacco products, increase the effectiveness of health warnings and prevent the design features of the pack from reducing the impact of prescribed government warnings.The Australian legislation was preceded by a series of evidence-based initiatives and was followed up with similar efforts after the implementation of the law, including an amendment to the domestic trademark law, to ensure successful implementation of the plain packaging law in the country. [bib_ref] Plainly constitutional: the upholding of plain tobacco packaging by the High Court..., Liberman [/bib_ref] IndIA-AusTrAlIA reseArCH CollAborATIon on PlAIn PACkAgIng While Australia implemented plain packaging as a part of the comprehensive tobacco control effort, experts in tobacco control from Australian and Indian institutions collaborated to form a joint India-Australia high-level taskforce to explore plain packaging in India. The taskforce produced a comprehensive report,and a research paper on the feasibility of plain packaging in India. [bib_ref] Exploring perception of indians about plain packaging of tobacco products: a mixed..., Arora [/bib_ref] However, the taskforce had concluded that further research was required before such policy measures could be introduced. In the meantime, a Private Member's Bill was introduced in the Indian Parliament seeking amendment to the COTPA to include plain packaging of tobacco products.The legality of plain packaging under international laws has been established with the Australian experience and as noted by experts. Indeed, India has its own legal and constitutional framework distinct from Australia and any other country's experience in introducing and implementing plain packaging. Moreover, tobacco control measures often become the subject of litigation in Indian courts and legal barriers have been perceived as a threat to the realisation of plain packaging.
## Who fctc recommends plain packaging
The WHO FCTC prohibits any kind of advertisement of tobacco products and recommends ensuring clear health warnings on tobacco products.Article 11 of WHO FCTC requires parties to implement large health warnings on tobacco packages to increase public awareness about the dangers of tobacco use.Going a step ahead, the guidelines on Article 11 (packaging and labelling),and Article 13 (tobacco advertising, promotion and sponsorship), 7 adopted by the Conference of the Parties in 2010, recommend that parties should consider adopting plain packaging. The Article 11 guidelines recommend that parties should consider adopting measures to restrict or prohibit the use of logos, colours, brand images or promotional information on packaging other than brand names and product names displayed in a standard colour and font style.FCTC oblIgATIons 'neCessAry', 'FAIr And equITAble' Although plain packaging is not expressly mentioned in the FCTC text, it has been clearly recommended under the guidelines for implementation of Articles 11 and 13. This makes plain packaging of tobacco products a necessary obligation for parties to FCTC. Furthermore, while dealing with the tobacco industry's challenge against larger graphic warning provisions in Uruguay, the tribunal held that the measures were based on and were in furtherance of the obligations and recommendations of the FCTC and thus not arbitrary, and do not violate the 'fair and equitable' treatment clause of the bilateral trade and investment treaties.PICTorIAl HeAlTH WArnIngs In IndIA India was among the forerunners in FCTC negotiations and was among the first few nations to ratify the treaty and simultaneously adopt a national tobacco control legislation in 2003. Even before the national legislation came into force, the Courts in India had given directives to the state machinery to take strict measures to curb the tobacco epidemic by taking into consideration the constitutional duty of the state to protect public health. Consistent with the mandates of the FCTC, the Indian law also prohibited sale and import of any tobacco BMJ Global Health products without the specified health warnings on the tobacco packages.However, what followed was an unprecedented delay and dilution of the pictorial health warnings notified initially, which were implemented on all tobacco packs in the country 5 years later, from June 2009. [bib_ref] Pictorial health warnings on tobacco products in India: sociopolitical and legal developments, Arora [/bib_ref] Since 2009, India implemented pictorial health warnings covering 40% of the front panel of the pack (ie, 20% of the principal display area of the pack) with the health warnings rotated every 2 years.These inadequate warnings placed India at 138th position for size of health warnings.However, on 1 April 2016, India implemented the much-awaited larger health warnings that cover 85% of the tobacco packs on both sides with 60% pictorial and 25% textual warning.The only other countries in the world with larger warnings are Nepal and Vanuatu at 90% and the other countries to equate such large graphic health warnings are Thailand (85%) followed by Australia at 82% and Uruguay as well as Sri Lanka at 80%.One of the most significant aspects of the new regulation is that it provides for a minimum size for the warnings, that is, not be <3.5 cm (width) and 4 cm (height) so as to ensure that the warnings are legible, prominent and conspicuous.THe logICAl nexT sTeP For TobACCo ConTrol PolICy In IndIA Adoption of the larger pictorial health warnings followed the recommendations made to Ministry of Health and Family Welfare, Government of India, by an expert committee constituted earlier in 2014 to look at global best practices and develop field tested India-specific pictorial health warnings.Although the warnings notified in October 2014 were to come into force from 1 April 2015, they were delayed owing to an observation from the Committee on Subordinate Legislation of the Lok Sabha (Lower House of Indian Parliament). Later, due to strict direction from the Rajasthan High Court,it came into force from 1 April 2016. The Court also referred to 'plain packaging as an improved and effective strategy, and therefore, suggested that it should be given a serious thought by legislature'.The issue of plain packaging was specifically raised in and addressed by the Allahabad High Court, wherein the court directed the Centre and the State Governments to consider implementation of plain packaging of tobacco products and observed that, 'tobacco plain packaging measure would be a long-term investment to safeguard the health of the Indian youth'. [bib_ref] Love Care Foundation v. Union of India and Others, Tobacco Free Kids [/bib_ref] The court strongly recommended the Indian government to to gather further evidence in support of plain packaging and implement plain packaging laws within the current tobacco control legislation in the country. The Report of the India-Australia Taskforce on Tobacco Control had already outlined the public interest and also the public support for plain packaging as a policy intervention in India and recommended that India could implement plain packaging with some amendments in COTPA. This recommendation made immediate way to the Parliament, when an amendment to sections 3, 5 and 7 of COTPA was introduced through a Private Member's Bill in the Parliament.The Bill proposes an increase in the size of the pictorial health warnings and prohibition on advertisements of tobacco products 'in' and 'on' packs and at point of sale by repealing the two provisos to section 5 of COTPA. Section 3 of the Bill proposes insertion of the following section 7A, specifically on plain packaging.
7A. Every package of cigarette or any other tobacco products shall comply with following requirements: a. No business name or trademark or any other mark shall appear anywhere on the package, other than as permitted under this Act. b. The business name or trademark or any other mark may appear on the package subject to following conditions: A. it shall appear only once on the outer surface of the package; B. it shall appear in one line only; C. it shall appear horizontally below the specified warning with the font style and font size as may be prescribed. c. The outer and inner surfaces of the package shall be of a specified colour and texture as may be prescribed. d. The outer and inner surfaces of the package shall not have any embossing, ridges or any irregularities of shape or size, or any other embellishments. e. The package shall not have any of the following features: A. it shall not reveal any text or picture when scratched; B. it shall not be made of such ink that appears or changes colour with passage of time; C. it shall not be made of such ink that appear visible in certain light; D. it shall not have any removable or folding tabs.
## India's preparedness compared with australia and the uk
The taskforce also suggested several other immediate, intermediate and long-term measures towards preparation and adoption of plain packaging in India, the foremost being the need to compile a body of evidence supporting plain packaging in the Indian context. The trajectory of Australian and the UK's experience, from preparation to the enactment and implementation of its legislation on plain packaging and the early outcomes compared with the developments in India (table 1) could provide an indication of India's preparedness before introducing this big-ticket tobacco control measure in the country.
## Probable barriers to plain packaging in india
Tobacco industry will be the biggest obstruction in the road to plain packaging in India: the industry has used intimidation to resist plain packaging in Australia,the UK and other countries. The tobacco industry in India will likely BMJ Global Health initiate spurious cases of violation of constitutional rights including freedom to trade, loss of livelihood, right to property and other laws including competition and consumer protection laws. Similarly, the tobacco industry in India has strongly resisted efforts to strengthen the pictorial health warnings and the 85% pack warnings are currently under judicial review.These tactics have resulted in a delay and dilution of pictorial health warnings in India.
Provisions under multilateral and bilateral trade and investment treaties are being used by the tobacco industry against plain packaging efforts: tobacco companies in Australia, the UK and elsewhere have exploited investment protections within international investment agreements to challenge plain packaging. [bib_ref] Time to Quit? Assessing international investment claims against plain tobacco packaging in..., Voon [/bib_ref] Tobacco companies claim that these laws violate trade and investment treaties and may result in the countries liability in huge compensation to the companies under the investor-state dispute settlement (ISDS).This insidious intimidation by the tobacco industry stands exposed in the claims filed against Uruguay's warning labels and branding measures and against Australia's plain packaging before Australia-Hong Kong ISDS and the dispute at the World Trade Organisation (WTO). However, legal experts suggest that plain packaging does not contravene a country's obligations under investment agreements or trade treaties and that public health has to be given prime importance.The importance of public health and the right of states to take measures aimed at protecting it are recognised across the international trade and investment regimes as 'both vital and important in the highest degree'.Plain packaging is consistent with the WTO norms: within the WTO regime, General Agreement on Tariffs and Trade Article XX(b) guarantees the Members' right to take steps necessary to protect the health of humans, animals and plants.While Article XIV(b) of the General Agreement on Trade in Services authorises Members to take measures for the protection of human, animal or plant life or health,and TRIPS Article 8.1 sets out the basic principle relating to health and allows Members to take measures for protecting public health.To make use of the health exceptions, WTO Agreements generally require the health measures be no more trade-restrictive than necessary.With respect to tobacco control, restrictions called for by the provisions in the FCTC may well be determined to be 'necessary' for health protection under the WTO rules.Unlike the tax increases and education programmes, plain packaging restricts the capacity of the tobacco packs to act as moving billboards. It aims to decrease consumers being attracted to appealing packs, which can be prevented only through standardised packing. Therefore, plain packaging, per se does not violate the WTO agreements including TRIPS agreement, and is 'necessary' and there is no 'alternative measure' that can achieve the desired objective. The Doha Declaration on TRIPS,and Public Health and the Punta del Este Declaration,further support this claim.
Overlapping laws and regulations: a lack of careful analysis of the legal and constitutional concerns with overlapping laws and regulations in India was identified as a barrier by the taskforce. These concerns have been summarised in table 2 along with a careful assessment mitigating the same.
## Countering the tobacco industry claims
The Australian and the UK's experience of implementing plain packaging of tobacco products makes it clear that the tobacco industry will remain the single largest barrier to implementation of an effective tobacco control measure such as plain packaging. However, the tobacco [fig_ref] Table 1: The plain packaging trajectory for Australia, the UK and India S [/fig_ref]
## Bmj global health
industry's challenge against plain packaging is not sustainable on constitutional or national and international legal or treaty regime. Their claims before the Australian Constitutional Court, United Kingdom Court of Appeal and the ISDS have all been rejected. According to legal experts, the dispute at the WTO against the Australian plain packaging decision has little chance of success, [bib_ref] Plainly constitutional: the upholding of plain tobacco packaging by the High Court..., Liberman [/bib_ref] and has already been decided in favour of Australia.Given the global attention and appetite for this significant tobacco control measure, WHO and the Secretariat for WHO FCTC has called on countries to get ready for the plain (standardised) packaging of tobacco products. Another concern against adopting plain packaging emanates from the tobacco industry generated apprehensions related to violation of trade and investment agreements and to avoid claims of intellectual property rights infringement. However, a closer examination of the WTO agreements with given health exceptions, the Doha and Punta Del Este Declarations on public health and decisions of the WTO and other dispute settlement tribunals suggest that a country could successfully implement a plain packaging regulation if it is applied in a non-discriminatory manner. Moreover, both domestic and international laws regulating trademarks do not give any absolute right of use over a registered trademark. It merely confers a negative right against the use of that trademark by others.
Parties to the FCTC must be mindful of the fact that, it is not that only the WTO agreements are binding; parties must respect their obligation under the WHO FCTC Articles 11 and 13, which stipulate implementation of effective packaging and labelling of tobacco products, and prohibition of tobacco advertisement, promotion and sponsorships. FCTC recommends plain packaging as an effective means to implement the two treaty provisions. Even the Australian High Court while ruling in favour of the plain packaging regulations, highlighted the implication of being a party to WHO FCTC and the need to give effect to obligations thereunder. [bib_ref] JT International SA v Commonwealth: tobacco plain packaging, Fletcher [/bib_ref] Moreover, the doctrine of Pacta sunt servanda contained in article 26 and article 27 of the Vienna Convention, 1980 lays down that every treaty in force is binding on the parties to it and must be performed in good faith and a party may not invoke the provisions of its internal law as a justification for its failure to perform a treaty obligation.It is also settled that when there is no conflict between Property in this respect is vested with no more than a negative right.
Competition Act Plain packaging will be trade restrictive and anticompetitive
As per section 54 of the Act, the Central Government has power to exempt the application of the very Act.
There is constitutional protection to take such action under Article 19.
## Consumer protection act
Plain packaging will prevent customers from the basic information about the product Plain packaging will reduce possibilities for 'unfair trade practice' by tobacco producers and manufacturers.
Consumer protection law is to be read in addition to and not in derogation of the provisions of COTPA. Legal Metrology Act Plain packaging will affect mandatory declarations on prepackaged commodities
Most of the mandatory declarations under this law are same as notified for tobacco products under COTPA regulations.
Law already exempts bidi manufacturers on certain declarations. BMJ Global Health the domestic law and a treaty obligation, Government instrumentalities should respect and implement the treaty obligations.
# Conclusion
The developments related to implementation of larger pictorial health warnings indicate that India is at a ripe stage from where, if supported by the strong political will, plain packaging will be the next obvious and logical step in the armoury of tobacco control. It is expected that India will have to face unique challenge from the unregulated markets of bidi and smokeless forms of tobacco, sale of loose tobacco products and a myriad of tobacco product varieties, most of which are produced and marketed in the unorganised sector. However, with strategic planning and preparedness, such challenges could be avoided when advancing the plain packaging agenda. This may be feasible through ensuring provisions in the official draft amendment to COTPA,and second, supporting the Private Member's bill on the amendment to COTPA. In addition to the constitutional and legal backing for a public health policy like plain packaging of tobacco products, the overwhelming support of the general public provides the much-needed impetus for its consideration by the law makers. [bib_ref] Perceptions and impact of plain packaging of tobacco products in low and..., Hughes [/bib_ref] Given India's 85% health warnings take a step closer to plain packaging and leaving not much space for tobacco industry to enhance appeal of the pack, plain packaging would still be useful as it will help in standardisation of packages across the myriad varieties of tobacco products available in India. At the risk of stating the obvious, provisions of WHO FCTC and the guidelines recommending plain packaging of tobacco products are not in conflict with any of the domestic laws of India, and rather complement COTPA. Considering the fact that several countries have already adopted plain packaging legislation, India, being a frontrunner in implementing stronger tobacco control measures, should also take the bold step before it is left far behind other countries.
[table] Table 1: The plain packaging trajectory for Australia, the UK and India S. no. [/table]
[table] Table 2: Assessment of key legislative and regulatory concernsThis freedom is not absolute and Article 19(6) of the Constitution provides that reasonable restrictions on trade may be imposed for protecting interest of the general public and may restrict any trade and activity even to the exclusion of all and also impose a total prohibition on such trade.Total prohibition of tobacco trade is possible while plain packaging is no more than a reasonable restriction in the interest of public. [/table]
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The Importance of Recognizing Diffuse Idiopathic Skeletal Hyperostosis for Neurosurgeons: A Review
Diffuse idiopathic skeletal hyperostosis (DISH) is characterized by calcification and ossification of the soft tissues, mainly ligaments and entheses. The spines of patients with DISH generally become increasingly rigid and osteoporotic, and fractures may occur after even a relatively minor traumatic event such as a ground-level fall. Moreover, the prevalence of DISH may be rapidly increasing in affluent societies. Thus, awareness of this condition is becoming more important for neurosurgeons when assessing trauma patients. For the present article, a literature review was conducted to summarize the current clinical, pathogenetic, and therapeutic knowledge of this disease. Furthermore, current treatment strategies for DISH-related spine injuries are also reviewed. Although the recommended treatment for spinal injuries in DISH patients is surgical, mainly through long-segment posterior fusion, rather than conservative options, stable fractures without any associated neurologic deficits have often been successfully managed with immobilization alone. Percutaneous instrumentation and the use of teriparatide may be useful depending on the surgical risks and patient neurological status. key words: spinal fracture, diffuse idiopathic skeletal hyperostosis, trauma, surgery Address reprint requests to: Masatoshi Yunoki, Md, department of neurosurgery, kagawa rosai Hospital,
# Introduction
diffuse idiopathic skeletal hyperostosis (diSH) is an idiopathic form of degenerative arthritis that typically affects males over 60 years of age. [bib_ref] Senile ankylosing hyperostosis of the spine, Forestier [/bib_ref] [bib_ref] Ankylosed spines are prone to fracture, Hunter T [/bib_ref] [bib_ref] Levine AM: Fractures of the spine in diffuse idiopathic skeletal hyperostosis, Paley D [/bib_ref] [bib_ref] Spinal fractures in patients with ankylosing spinal disorders: a systematic review of..., Westerveld [/bib_ref] [bib_ref] Vaccaro Ar: the management of spinal injuries in patients with ankylosing spondylitis..., Whang [/bib_ref] diSH is characterized by exuberant bony growth along the anterior longitudinal ligament. 1) because the condition is usually painless and latent, diSH is often incidentally diagnosed by radiographic examination taken for a non-related reason. diSH causes ankylosis of the spinal column, which can make the spine prone to fracture after even trivial trauma. Moreover, such unstable fractures can lead to secondary neurologic deterioration in initially asymptomatic patients. [bib_ref] Ankylosed spines are prone to fracture, Hunter T [/bib_ref] [bib_ref] Levine AM: Fractures of the spine in diffuse idiopathic skeletal hyperostosis, Paley D [/bib_ref] [bib_ref] Spinal fractures in patients with ankylosing spinal disorders: a systematic review of..., Westerveld [/bib_ref] [bib_ref] Vaccaro Ar: the management of spinal injuries in patients with ankylosing spondylitis..., Whang [/bib_ref] the prevalence of diSH may be rapidly increasing in affluent societies. thus, awareness of this condition should be increased among neurosurgeons that assess trauma patients and treat spinal injuries. [bib_ref] Spinal fractures in patients with ankylosing spinal disorders: a systematic review of..., Westerveld [/bib_ref] in the present article, a review of the literature was conducted to summarize the current clinical, pathogenetic, and therapeutic knowledge of this disease. in addition, the current treatment strategies for diSH-related spine injuries are also reviewed.
## Disease characteristics and diagnosis of dish
diSH, then termed senile ankylosing hyperostosis, was first described by Forestier and rotes-Querol more than 50 years ago. 1) Later, because a similar condition was also recognized in younger patients, the name ankylosing spinal hyperostosis (ASH) was adopted. [bib_ref] Ankylosing hyperostosis of the spine, Forestier [/bib_ref] As the involvement of the axial skeleton, particularly the thoracic spine and the peripheral joints were recognized, researchers began using the term diSH. 8) the disease mainly targets the enthesis, an organ that is rich in collagen fibers, fibroblasts, other mesenchymal cells, fibrocartilage, and calcified matrix that penetrates the bone cortex at its attachment. Although there are no validated diagnostic criteria for diSH, three sets of criteria are often used for diagnosis. the most commonly used classification criteria were defined by resnick and niwayama. these criteria include the involvement of at least four contiguous vertebrae of the thoracic spine, preservation of the intervertebral disc space, and the absence of apophyseal joints and sacroiliac inflammatory changes. [bib_ref] radiographic and pathologic features of spinal involvement in diffuse idiopathic skeletal hyperostosis..., Resnick D, Niwayama [/bib_ref]. there was an increase in the prevalence of diSH with increasing age from 1% in the 40-49-year-age group to 13.6% in those over 70 years. 13) Furthermore, Weinfeld et al. analyzed data from two large American midwest metropolitan hospital populations to conclude that the prevalence of diSH in that mostly Anglo-Saxon population over 50 years of age was 25% in males and 15% in females. At 70 years of age, furthermore, the prevalence in males and females increased to 35% and 26%. 14) based on these data, diSH is likely to be more frequent and more severe in men throughout life, and the incidence of the disease increases with age. Furthermore, a higher prevalence was reported in Caucasian populations than in korean or black Africans. [bib_ref] the prevalence of diffuse idiopathic skeletal hyperostosis in African blacks, Cassim B [/bib_ref] [bib_ref] Jb: the prevalence of diffuse idiopathic skeletal hyperostosis in korea, Kim Sk [/bib_ref] thus, ethnic factors are also likely important factors in the prevalence of diSH. the etiology of diSH also remains unclear. However, several metabolic conditions (obesity, diabetes mellitus, hypertension, hyperinsulinemia, and dyslipidemia, among others), genetic factors human leukocyte antigen (HLA) and collagen type Vi alpha 1 (CoL6A1) genes, environmental exposures (fluoride, vitamin A/retinol), and drugs (isotretinoin, etretinate, acitretin, and other vitamin A derivatives) are reported to be associated with diSH. [bib_ref] Senile ankylosing hyperostosis of the spine, Forestier [/bib_ref] [bib_ref] Ankylosed spines are prone to fracture, Hunter T [/bib_ref] [bib_ref] Levine AM: Fractures of the spine in diffuse idiopathic skeletal hyperostosis, Paley D [/bib_ref] [bib_ref] Spinal fractures in patients with ankylosing spinal disorders: a systematic review of..., Westerveld [/bib_ref] [bib_ref] Vaccaro Ar: the management of spinal injuries in patients with ankylosing spondylitis..., Whang [/bib_ref] in addition, two recent studies showed that patients with diSH have a higher incidence of risk factors for stroke, a higher prevalence of metabolic syndrome, and a higher risk for future coronary events. thus, they are encouraged to take measures to reduce the risk of such complications. [bib_ref] Metabolic syndrome and cardiovascular risk in patients with diffuse idiopathic skeletal hyperostosis, Mader R [/bib_ref] [bib_ref] diffuse idiopathic skeletal hyperostosis associated with risk factors for stroke: a case-control..., Miyazawa N [/bib_ref] sites on the thoracic spine, which are also reported to be involved in diSH. [bib_ref] the epidemiology of hyperostosis of the spine together with its symptoms and..., Julkunen [/bib_ref] neither of these two sets of criteria assess the peripheral manifestations of the condition. However, the criteria for diSH defined by utsinger et al. lowered the threshold for spinal involvement to three contiguous vertebral bodies, but added the presence of peripheral enthesopathies to the diagnostic measures (table 1). [bib_ref] Shapiro r: diffuse skeletal abnormalities in Forestier disease, Utsinger Pd [/bib_ref] the differential diagnosis of diSH requires distinguishing it from ankylosing spondylitis (AS). An AS is a relatively rare disease with a prevalence of 0.05-1.4%, whereas the diSH has a prevalence ranging from 2.9% to 25%. [bib_ref] Spinal fractures in patients with ankylosing spinal disorders: a systematic review of..., Westerveld [/bib_ref] [bib_ref] Ankylosing spondylitis: a state of the art factual backbone, Ghasemi-Rad [/bib_ref] it is a chronic inflammatory rheumatic disease that tends to affect relatively young white males. [bib_ref] Spinal fractures in patients with ankylosing spinal disorders: a systematic review of..., Westerveld [/bib_ref] [bib_ref] Ankylosing spondylitis: a state of the art factual backbone, Ghasemi-Rad [/bib_ref] Patients with AS are usually symptomatic, but suffer from a myriad of associated conditions such as iritis, uveitis, or ulcerative colitis. Sacroiliac and apophyseal fusion or sclerosis, the earliest symptoms of which are back pain and stiffness, result from inflammation of the sacroiliac joint. [bib_ref] Ankylosing spondylitis: a state of the art factual backbone, Ghasemi-Rad [/bib_ref] this inflammation can gradually spread to the joints between the vertebrae, causing a condition called spondylitis, as well as to other joints including the
## Etiology and diagnosis of spinal fracture in dish patients
because diSH exposes injury sites to larger moments via the lever arms produced by stiffness of the otherwise largely ankylosed spinal column, even harmless-appearing fractures can become highly unstable and place the patient at high risk for a neurologic injury, particularly if the cervical segment is involved. [bib_ref] Ankylosed spines are prone to fracture, Hunter T [/bib_ref] [bib_ref] Levine AM: Fractures of the spine in diffuse idiopathic skeletal hyperostosis, Paley D [/bib_ref] [bib_ref] Spinal fractures in patients with ankylosing spinal disorders: a systematic review of..., Westerveld [/bib_ref] [bib_ref] Vaccaro Ar: the management of spinal injuries in patients with ankylosing spondylitis..., Whang [/bib_ref] [bib_ref] Cervical spine trauma in diffuse idiopathic skeletal hyperostosis: injury characteristics and outcome..., Koller [/bib_ref] Previous studies have shown that these fractures most commonly occur in the cervical spine, followed by the thoracic and lumbar segments. [bib_ref] Spinal fractures in patients with ankylosing spinal disorders: a systematic review of..., Westerveld [/bib_ref] , and flexion-type fractures in none of the diSH patients. 5) in addition, a higher incidence of missed fractures in these patients have been reported to led to the higher rates of spinal cord injury. [bib_ref] Ankylosed spines are prone to fracture, Hunter T [/bib_ref] [bib_ref] Levine AM: Fractures of the spine in diffuse idiopathic skeletal hyperostosis, Paley D [/bib_ref] [bib_ref] Spinal fractures in patients with ankylosing spinal disorders: a systematic review of..., Westerveld [/bib_ref] [bib_ref] Vaccaro Ar: the management of spinal injuries in patients with ankylosing spondylitis..., Whang [/bib_ref] [bib_ref] Cervical spine trauma in diffuse idiopathic skeletal hyperostosis: injury characteristics and outcome..., Koller [/bib_ref] in diSH-related spine fractures, delayed diagnosis is reported to be particularly common in nondisplaced fractures after trivial injuries and was associated with neurologic worsening.because of the difficulty in radiographically visualizing spines affected by diSH, screening of the entire spinal column with an advanced neuroimaging modality (magnetic resonance imaging or computed tomography) has been recommended especially for patients with persistent neck or back pain. [bib_ref] Vaccaro Ar: the management of spinal injuries in patients with ankylosing spondylitis..., Whang [/bib_ref] in Japan, there are many facilities where trauma patients whose main injury site is the head are treated predominantly by neurosurgeons. especially when head trauma is accompanied by an intracranial hemorrhage (iCH), patients with even mild symptoms are admitted directly to the department of neurosurgery to avoid delayed deterioration. [bib_ref] Clinical characteristics and problems of traumatic brain injury in the elderly, Hayashi T [/bib_ref] during the initial management of traumatic iCH, the diagnosis of spinal fracture is difficult because of an altered level of consciousness [fig_ref] Figure 1 A: case of cervical spinal fracture with diffuse idiopathic skeletal hyperostosis [/fig_ref]. in addition, symptoms recent report described the efficacy of teriparatide (tPtd) treatment for spine fractures in patients with diSH. [bib_ref] Successful treatment of spine fracture for diffuse idiopathic skeletal hyperostosis with teriparatide-a..., Iida [/bib_ref] beyond the use for fresh fractures, the successful treatment of a lumbar spine fracture with delayed union in a patient with diSH using daily administration of tPtd was also reported. [bib_ref] effective treatment of delayed union of a lumbar vertebral fracture with daily..., Matsumoto T [/bib_ref] in cases where surgical treatment is not indicated because of poor general condition, tPtd treatment is one option.
## Surgical procedure
A variety of surgical approaches have been reported to treat fractures in patients with diSH, including anterior, posterior, and combined approaches. However the most common approach based on the published literature is posterior instrumentation. [bib_ref] Levine AM: Fractures of the spine in diffuse idiopathic skeletal hyperostosis, Paley D [/bib_ref] [bib_ref] Spinal fractures in patients with ankylosing spinal disorders: a systematic review of..., Westerveld [/bib_ref] [bib_ref] Vaccaro Ar: the management of spinal injuries in patients with ankylosing spondylitis..., Whang [/bib_ref] [bib_ref] Cervical spine trauma in diffuse idiopathic skeletal hyperostosis: injury characteristics and outcome..., Koller [/bib_ref] because of the torque associated with the lever arms in conjunction with poor, osteoporotic bone quality, posterior surgery often requires multiple points of pedicle screw fixation above and below the affected level to appropriately stabilize the spinal column and heal the fracture. [bib_ref] Levine AM: Fractures of the spine in diffuse idiopathic skeletal hyperostosis, Paley D [/bib_ref] [bib_ref] Spinal fractures in patients with ankylosing spinal disorders: a systematic review of..., Westerveld [/bib_ref] [bib_ref] Vaccaro Ar: the management of spinal injuries in patients with ankylosing spondylitis..., Whang [/bib_ref] [bib_ref] Cervical spine trauma in diffuse idiopathic skeletal hyperostosis: injury characteristics and outcome..., Koller [/bib_ref] Most reported case series on surgery for fractures in diSH patients were based on traditional open procedures with pedicle screw placement and the creation of a fusion bed. [bib_ref] Senile ankylosing hyperostosis of the spine, Forestier [/bib_ref] [bib_ref] the epidemiology of hyperostosis of the spine together with its symptoms and..., Julkunen [/bib_ref] [bib_ref] osteoporotic Fractures in Men (MroS) Study Group: diffuse idiopathic skeletal hyperostosis and..., Holton Kf, Denard [/bib_ref] [bib_ref] the prevalence of diffuse idiopathic skeletal hyperostosis in African blacks, Cassim B [/bib_ref] [bib_ref] Jb: the prevalence of diffuse idiopathic skeletal hyperostosis in korea, Kim Sk [/bib_ref] However, percutaneous instrumentation has become more popular for the associated with spinal fracture in diSH patients may be minor . neurosurgeons therefore must be careful to remain aware of the possible presence of diSH in these patients.
## Treatment of spinal fractures in dish patients
treatment for ankylosed spine fractures in diSH patients may be operative or nonoperative depending on the patient age, medical comorbidities, fracture pattern, and neurologic status. Conservative treatment is usually performed by bracing and bed rest. However, surgical rather than conservative treatment is generally recommended based on the incidence of complications and the prognosis of such fractures. [bib_ref] Spinal fractures in patients with ankylosing spinal disorders: a systematic review of..., Westerveld [/bib_ref] treatment of thoracolumbar trauma, [bib_ref] Surgical treatment of traumatic fractures of the thoracic and lumbar spine: a..., Verlaan [/bib_ref] though this approach has been criticized for the lack of data regarding the rates of pseudarthrosis and hardware failure when used for fractures in diSH patients. recently, nayak et al. reported that percutaneous instrumentation can be successfully used to manage spinal fractures in patients with diSH while preserving a good postoperative quality-of-life with limited disability when used on patients with good preoperative neurologic status. [bib_ref] Minimally invasive surgery for traumatic fractures in ankylosing spinal diseases, Nayak Nr [/bib_ref] Conclusion recognition of diSH is important for neurosurgeons when assessing trauma patients because this condition can make the spine prone to fracture after even trivial trauma, which frequently results in delayed diagnosis and neurologic deterioration. Although the current recommendation is for these spinal injuries to be treated surgically, mainly through long-segment posterior fusion, rather than conservatively, stable fractures without any associated neurologic deficits have often been successfully managed with immobilization alone. Percutaneous instrumentation and the use of tPtd are other options depending on the risk of surgery and patient neurological status.
[fig] Figure 1 A: case of cervical spinal fracture with diffuse idiopathic skeletal hyperostosis (DISH) accompanying traumatic subarachnoid hemorrhage. After falling from a low height, a 79-year-old man presented with a laceration of the forehead. His consciousness level was 300 (JCS). He was admitted to the Department of Neurosurgery because head CT and MRI scans revealed traumatic subarachnoid hemorrhage (A). MRI and MR angiography detected pontine infarction and decreased blood flow in the vertebrobasilar artery (B, C). Cervical CT detected a small crack (arrow) in the anterior osteophyte of C4/5 in the setting of ankylosed spine due to DISH (D). Dynamic cervical spine XP showed an extension fracture through the C4/5 disc space (E, F). After 30 days of conservative treatment to stabilize his vital signs, he underwent posterior fixation surgery (G, H). [/fig]
[table] Table 1: Diagnostic criteria for diffuse idiopathic skeletal hyperostosis [/table]
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Emerging Treatments for Malignant Pleural Mesothelioma: Where Are We Heading?
Malignant pleural mesothelioma (MPM) is an uncommon but aggressive and treatment resistant neoplasm with low survival rates. In the last years we assisted to an exponential growth in the appreciation of mesothelioma pathobiology, leading several new treatments to be investigated both in the early stage of the disease and in the advanced setting. In particular, expectations are now high that immunotherapy will have a leading role in the next years. However, caution is required as results from phase II studies in MPM were often not replicated in larger, randomized, phase III trials. In this review, we describe the most promising emerging therapies for the treatment of MPM, discussing the biological rationale underlying their development as well as the issues surrounding clinical trial design and proper selection of patients for every treatment.
# Introduction
Malignant pleural mesothelioma (MPM) is an uncommon and highly lethal cancer. The annual incidence of MPM ranges between 10 cases per million to 29 cases per million depending on the country and, because of the long latency period, the peak is expected in the 2020s (1) in high-income countries. In addition, according to WHO prediction, developing countries where asbestos is still used, are likely to face a new epidemic of asbestos-related diseases, including MPM.
MPM pathogenesis is peculiar, as the direct causal relationship between exposure to airborne asbestos particles and the development of MPM is well established. The chronic exposure to asbestos fibers, which may enter the lung periphery and the pleura, leads to chronic inflammation of the mesothelium which sustains the carcinogenic processes. Individuals with germline BRCA1 associated protein-1 (BAP1) mutations may be predisposed to MPM, since they may develop it without any apparent asbestos exposure. Recent biological and preclinical studies provided further insights into MPM carcinogenesis, revealing the importance of tumor suppressor gene inactivation, through several mechanisms (single nucleotide variants (SNVs), copy number losses, gene fusions, and splicing alterations). Tumor suppressor genes highly altered are cyclin-dependent kinase inhibitor 2A (CDKN2A, 60% of the cases), BAP1 (60% of the cases also in sporadic MPM), and neurofibromin 2 (NF2, 75% of the cases).
The chronic inflammatory response to asbestos involved in the pathogenesis of MPM also causes a unique tumor environment. This microenvironment is mainly composed of immunosuppressive cells [regulatory T cells, macrophages and myeloid-derived suppressor cells (MDSCs)] and the number of these cells as determined by immunohistochemistry (IHC) represents a negative prognostic factor. On the other hand, immuneactivating responses, such as the presence of CD8 + T cells, are correlated with better outcome, although such links with prognosis are less important when compared with other cancer entities which are more immunogenic than MPM (12).
The management of MPM is complex and outcomes remain poor. For patients with early stage MPM the role of radical surgery is still a matter of debate and it should be considered only as part of a multimodal treatment (i.e., surgery combined with chemotherapy, radiotherapy, or both). Looking at unresectable MPM, no major breakthroughs have been made since the approval of antifolate and platinum combination chemotherapy. Median overall survival (OS) time with standard first-line options is about 13 months, with the best outcome for the epithelioid MPM subtype. Second-line treatment scenario is even more disappointing. With the only exception of a repeated course of pemetrexed-based chemotherapy for previously responsive patients, limited options are available for relapsed MPM and new treatments are urgently needed.
Steps have been made toward a best appreciation of mesothelioma biology and have been essential to identify novel molecular therapeutic targets, representing the rationale for testing multiple targeted therapies in MPM. Nevertheless, the potential to improve the potency and the specificity of the immune system, along with recent successes in other thoracic tumors, have attracted a growing interest in cancer immunotherapy. Continue efforts are necessary to further deepen our understanding of mesothelioma, taking into account biological and temporal heterogeneity of the disease in order to finally optimize the development of new treatment options in the context of well-designed clinical trials.
In this review, we describe last emerging therapies for mesothelioma, discussing the current status of knowledge in mesothelioma genetics and immune-biology, as well as the issues surrounding the conduction of high-quality trials in MPM and the selection of best patients for different treatments.
## Neoadjuvant/adjuvant setting
Due to the anatomy, microscopically radical (R0) resection is not achievable in mesothelioma surgery and the goal of mesothelioma surgery is macroscopic complete resection (R1). Surgery alone is not curative; it is usually performed with chemotherapy and/or radiation therapy and reserved to a subset of patients with early tumor stage, epithelioid histology and good performance status.
Therapeutic surgery in mesothelioma has historically involved either an extended pleurectomy-decortication (eP/D) or an extrapleural pneumonectomy (EPP). eP/D has been proven to offer better results in the context of multimodality treatment, and although the benefit of systemic therapy has been shown only in the advanced/unresectable disease, it is common practice to give four cycles of cisplatin or carboplatin with pemetrexed as adjuvant or neoadjuvant therapy. Two ongoing trials, MARS 2 (NCT02040272) and EORTC1205-LCG (NCT02436733), are currently evaluating the usefulness, the feasibility and the best timing for the combined approach of surgery and chemotherapy.
In order to improve local control and ideally survival, radiotherapy can be given. New approaches of radical hemithoracic radiation using intensity-modulated techniques are being tested. Rimner et al. showed that hemithoracic intensity-modulated pleural radiation therapy (IMPRINT) after chemotherapy and P/D was safe in 27 MPM patients as part of a multimodality lung-sparing treatment, with an acceptable rate of radiation pneumonitis. Larger clinical trials are awaited to confirm the effectiveness of this approach.
Recently, intrapleural therapies have been reported with the aim of improving loco-regional control of the disease by spreading drugs directly on the tumor surface. Several techniques with different rationale have been used with promising results: hypertermic intrapleural chemotherapy, photodynamic therapy (PDT), intrapleural immunotherapies [interferons (IFNs) and interleukin-2 (IL-2)], and gene therapy. However, available evidences are mainly based on retrospective, small and single-institution studies and controlled randomized trials are required.
If given as neoadjuvant therapy, novel agents should have the ability to induce tumor shrinkage, increasing the possibility of a complete microscopic resection and ultimately prolonging overall survival while maintaining a good safety profile. Designing studies in this setting remains a challenging effort that requires multidisciplinary involvement. Nevertheless, the neoadjuvant setting provides the unique possibility to conduct translational research in the context of window-ofopportunity trials, acquiring valuable information from blood and tissue collection. For example, the focal adhesion kinase (FAK)-inhibitor defactinib showed immunomodulatory effects when administered pre-operatively in a phase II window of opportunity trial (23) with a good tolerability profile, an objective response rate of 13% and 67% of stable disease, thus not altering resectability or mortality compared to historical controls. Final trial data are expected for 2020. This approach has also paved the way for testing the properties of immune check-point inhibitors (CIs). There are several ongoing neoadjuvant trials which aim to assess the immunomodulatory and pharmacodynamics effect of CIs, as monotherapy (NCT02707666), as combination of anticytotoxic T-lymphocyte-associated protein 4 (CTLA4) and antiprogrammed cell death protein (PD-1) agents (NCT02592551, NCT03918252) and as combination of anti-programmed deathligand 1 (PD-L1) with standard chemotherapy (NCT03228537).
By assessing translational surrogates of response, these trials may represent an opportunity to look into predictive biomarkers, improving selection of candidates to CIs-treatment.
CIs are also tested in the adjuvant setting (NCT02707666). From an immunological perspective, the main goal of combining surgery with adjuvant CIs is to reduce tumor induced immunosuppression. Increased tumor size correlates with major immune suppression and surgically shrinking tumor size may potentially reduce immune inhibition and T-cell exhaustion. Another approach to increase immune activation in the adjuvant setting is represented by vaccines, either protein, bacteria or cell-based. An adjuvant Wilms tumor 1 (WT1) vaccine (galinpepimut-S), given with granulocyte-macrophage colony-stimulating factor (GM-CSF) and an immunologic adjuvant called montanide ISA 51 UFCH in MPM patients whose tumors expressed WT1 at IHC, had completed combined multimodality therapy and had no evidence of disease, showed a median progression-free survival (PFS) of 10.1 months (95% CI 5.5-20.8 months) and a median OS of 22.8 months (95% CI 9.1-37.6 months) with a favorable safety profile. Galinpepimut-S is currently being tested in the advanced setting combined with CI-treatment (NCT04040231).
In peritoneal mesothelioma, the feasibility of administering dendritic cells pulsed with an allogenic tumor cell lysate after cytoreductive surgery and hyperthermic intraperitoneal chemotherapy (HIPEC) is being assessed in the ongoing MESOPEC trial (NTR7060). Secondary objectives of the study are to assess the safety of dendritic cells and determine whether this adjuvant treatment may induce a specific immunological response against the tumor. Pre-clinical evidences showed that dendritic cell therapy leads to better outcome when dendritic cells are injected in murine models with lower tumor volume. An efficient immune response is hampered by cytokines and regulatory T-cells induced by mesothelioma cells, showing that a low tumor load correlates with a better functioning immune system and higher anti-tumor responses. Giving dendritic cell therapy after surgically reducing tumor load might therefore improve response to therapy and clinical outcome.
To date, despite the neoadjuvant/adjuvant treatment represents a promising setting to test new therapeutic strategies, the global level of evidence is quite low and international guidelines (30) do not recommend either neoadjuvant or adjuvant radiotherapy/chemotherapy as standard options for resectable MPM.
## Unresectable mesothelioma chemotherapy
There is no approved maintenance treatment for MPM patients who did not progress after first-line chemotherapy. NVALT19 was an open label, multicentric, randomized phase II trial, in which patients were assigned 1:1 to gemcitabine (1,250 mg/m2 day 1 and 8 of 3 weekly schedule) or best supportive care (BSC) after 4-6 cycles of first-line platinum-pemetrexed without progression. Data presented at the last ESMO conference showed an improvement in PFS (median 6.2 months vs. 3.2 months in the BSC arm [hazard ratio (HR) 0.42 (95% CI 0.28-0.63), p < 0.0001)], at the cost of an increased yet manageable toxicity (57% of patients experienced grade 3-4 adverse events vs. 13% in the BSC-arm, with neutropenia, nausea and lung infection being the most frequent). Since post-study treatments and OS data were not reported, the reported improvement in PFS could be simply due to an anticipation of second-line therapy.
Lurbinectedin is a new molecule that binds to the DNA minor groove in regulatory regions, inhibiting the function of oncogenic transcription factors. It also modulates the transcriptional program of monocytes and TAMs, hampering cytokine production. Investigator tested the role of lurbinectedin in the context of relapsed MPM, where no approved therapy exists. Recent data from the SAKK 17/16 multi-center, single-arm phase II trial, showed activity of lurbinectedin. Median PFS and median OS were 4.1 months (95% CI 2.6-5.5) and 11.9 months (95% CI 9.2-14.7), respectively. Lurbinectedin also worked independently of histology or prior immunotherapy.
These data support evaluation of the both gemcitabine as switch maintenance and lurbinectedin as second-line strategy in larger, randomized, phase III trials.
The NovoTTF-100L represents another approach that has been recently investigated to improve the efficacy of chemotherapy. NovoTTF-100L is a portable Tumor Treating Fields (TTFields) delivery system. TTFields represent a noninvasive, regional treatment modality by which alternating electric fields (at a frequency of 150 kHz) are continuously administer to the local site to arrest tumor cancer cell division. In human mesothelioma cell cultures, combining TTFields with cisplatin or pemetrexed led to reduction in cell count, induction of apoptosis and reduced clonogenic potential. These alternating electric fields act by disrupting spindle formation during metaphase and blocking the localization of intracellular organelles during telophase.
Based on the results of the prospective, single-arm, phase II STELLAR trial, the NovoTTF-100L System was approved by U.S. FDA in combination with pemetrexed plus platinum-based chemotherapy for the first-line treatment of unresectable locally advanced or metastatic MPM. NovoTTF-100L was approved under Humanitarian Device Exemption, an approval process guaranteed by the U.S. FDA which, taking into consideration the urgent need to identify more effective treatments for rare disease (such as MPM), allows medical devices to be marketed without requiring evidence of effectiveness.
However, the STELLAR trial raised several issues that need to be addressed before implementing this strategy into daily practice. The 80 patients enrolled in the STELLAR trial (34) had a median OS of 18.2 months (95% CI 12.1-25.8), with 40.3% of partial responses and 97.2% of them obtaining a clinical benefit. Response rates were similar to the ones with standard chemotherapy but lasted longer by adding TTFields (median response duration was 5.7 months, ranging from 1.4 to 13 months). The rate of serious systemic adverse events remained the same when NovoTTF-100L was added to chemotherapy (either pemetrexed plus cisplatin or pemetrexed plus carboplatin, according to investigator choice). Expected TTFields-related skin toxicity was reported in 66% (53 patients) with only 5% of grade 3 skin toxicity. These results should be considered in context of the randomized phase III MAPS trial, in which bevacizumab added to pemetrexed and cisplatin significantly improved median OS compared to pemetrexed plus cisplatin alone (median OS 18.8 vs. 16.1 months, HR 0.77, p = 0.0167). The control arm of this trial performed 4 months better than the historical cohort analyzed by Ceresoli et al.-the landmark study by and should be considered while discussing STELLAR data. Also PFS (7.6 months) and response (40%) were similar when compared to control groups in the MAPS and the recent LUME-meso trials. This fact, together with the potential sampling bias in single-arm studies and the effect of subsequent therapies, limits the interpretation of STELLAR data.
To date, TTFields represent one of many empirical approaches to MMP and further investigation of this approach in randomized trials is strongly encouraged.
## Anti-angiogenic agents
Activation of the vascular endothelial growth factor (VEGF) pathway, via its tyrosine kinase receptors, is crucial for mesothelioma cells growth (37), thus representing a rationale for antiangiogenic treatments in this neoplasm.
The addition of bevacizumab to pemetrexed and cisplatin chemotherapy as first-line treatment with bevacizumab maintenance therapy in patients who did not progress showed improved overall survival. However, bevacizumab remains currently unlicensed in this setting since the MAPS trial was not a registration trial. Moreover, results of Bevacizumab [an anti-VEGF monoclonal antibody (mAb)] as first-line option in combination with chemotherapy were not confirmed by other anti-angiogenic agents, such as the tyrosine-kinase inhibitors (TKIs) axitinib (an anti-VEGFR TKI), sorafenib (anti-VEGFR2/3, platelet-derived growth factor receptor (PDGFR) and rapidly accelerated fibrosarcoma (RAF)/c-KIT), or imatinib mesylate (targeting BCR-ABL, c-KIT, and PDGFR).
Since the benefit in the phase 2 trial (n = 87 patients) (42) was higher in epithelioid MPM than in non-epithelioid subtypes, the multi-targeted anti-angiogenic kinase inhibitor, nintedanib (targeting VEGFR 1-3, PDGFR α or β, fibroblast growth factor receptor (FGFR) 1-3, SRC and ABL kinases pathways) was tested in conjunction with first-line cisplatin plus pemetrexed in a randomized phase III trial vs. placebo only in patients with epithelioid histology. However, among the 458 randomized patients, the previous phase II efficacy findings were not confirmed and PFS did not differ between the nintedanib group (median 6.8 months [95% CI 6.1-7.0)] and the placebo group (7.0 months (95% CI 6.7-7.2); HR 1.01 (95% CI 0.79-1.30), p = 0.91). The interim analysis of OS also showed no difference between groups.
Nintedanib is also being currently investigated as only maintenance treatment for patients non-progressive after first line chemotherapy (NCT02863055).
Cediranib, a VEGFR and PDGFR inhibitor, added to first-line platinum-based chemotherapy, improved PFS in a randomized phase II trial. Primary end-point of the trial was to detect a PFS difference (by RECIST version 1.1) at the 1-sided 0.10 level and it was met. PFS was significantly higher in MPM patients who received cisplatin-pemetrexed chemotherapy with cediranib followed by maintenance cediranib, compared to the ones receiving cisplatin-pemetrexed with placebo. HR was 0.69 (median PFS 7.2 vs. 5.6 months, p = 0.096). However, PFS was not different by modified RECIST and no significant difference in OS was reported. As with bevacizumab, cediranib is not approved as first-line treatment combined with chemotherapy.
Ramucirumab is a monoclonal antibody that binds the extracellular domain of human VEGFR-2. Due to VEGF-R2 expression on macrophages, ramucirumab also inhibits macrophages and their infiltration into mesothelioma microenvironment, thereby decreasing tumor growth and proliferation. One-hundred sixty-four patients are planned to be randomized in a multicenter, double-blind, placebocontrolled phase II trial comparing gemcitabine with or without ramucirumab in the second-line setting [NCT03560973 (RAMES)], whose completion is expected for 2020.
## Targeted therapies
New studies have recently provided a comprehensive genomic profiling of mesothelioma. Genomic analysis may help in detecting actionable alterations and developing more tailored and effective therapies for MPM patients. Tumor suppressor inactivation (loss-of-function) represents one of the most frequent mutational events in this tumor. In addition, multiple studies have pointed out frequent copy gains and copy losses involving different portions of the genome.
Carriers of inherited loss-of-function mutations in BAP1 are predisposed to mesothelioma. BAP1 encodes a deubiquitinase enzyme, a member of the ubiquitin carboxy (C)terminal hydrolase (UCH) family, involved in different cellular pathways among which the cell cycle, cellular differentiation, cell death, metabolism, and the DNA damage response. In particular, BAP1 is thought to bind to the breast cancer type 1 susceptibility protein (BRCA1) and the BRCA1-associated RING domain protein 1 (BARD1) and enhance their tumor suppressor function. Besides germline mutations, recent analysis of the BAP1 locus by targeted next-generation sequencing identified homozygous inactivating mutations in approximately 60% of patients. This implies that the role of BAP1 in defective DNA repair and homologous recombination might be therapeutically exploited in a large number of MPM.
In a recent paper, among 385 patients treated with platinum chemotherapy, median OS was increased for MPM patients who had inherited mutations in DNA repair and/or other tumor suppressor genes. This is consistent with what already observed in ovarian and breast cancer patients with inherited mutations in BRCA1 and BRCA2. Conversely, BAP1 mutant mesothelioma cell lines resulted significantly less sensitive than BAP1 wild type cells to gemcitabine. In addition, the role of somatic BAP1 expression in MPM patients receiving chemotherapy still represents a matter of debate, with retrospective studies showing contradictory evidences.
By inducing synthetic lethality of alternate DNA repair pathways, poly-ADP ribose polymerase (PARP) inhibitors have proved to be able to cause cell death in cell lines with loss of function of BAP1. This observation suggests that patients with mutations in BAP1 and in DNA repair genes might also benefit from treatment with PARP inhibitors. An enrolling clinical trial in MPM patients is examining the relationship between patient genotype and response to the PARP inhibitor olaparib (NCT03531840). Another PARP inhibitor, niraparib, is being tested in patients with BAP1 and other DNA damage response (DDR) pathway deficient neoplasms including mesothelioma (NCT03207347). BAP1 inactivation also works as a putative epigenetic regulator involved in the polycomb repressive complex 2 (PRC2) and enhancer of zeste-homolog 2 (EZH2) pathway. Mesotheliomas with BAP1 loss proved to be responsive to EZH2 inhibition in vitro and in vivo. EZH inhibition may then represent a promising strategy, with tazemetostat showing a promising disease control rate of 51% at 12 weeks in a multicenter phase 2 trial (64).
CDKN2A is a tumor suppressor gene frequently inactivated in mesothelioma. CDKN2A encodes the ADP-ribosylation factor (ARF, also known as p14) and INK4A (also known as p16) via alternative reading frames. By inhibiting cyclin-dependent kinase 4 (CDK4) and CDK6, INK4A decelerates the G1-S cell cycle transition. Small molecules CDK4 and CDK6 inhibitors induce apoptosis in CDKN2A-mutated tumorsand MPM cell lines viability was inhibited in a dosedependent manner by the CDK4/CDK6 inhibitor abemaciclib. Combined with radiotherapy, this agent also completely suppressed tumor growth in a mouse model of MPM. These finding led to the investigation of abemaciclib in p16INK4A negative MPM patients [NCT03654833 (MiST)].
The hepatocyte growth factor (HGF), by binding to the MET receptor and activating its downstream target PI3K has been shown to enhance MPM cell proliferation, migration and invasiveness. Therefore, this pathway represents a compelling therapeutic target in this disease. However, the modest response rate observed in the early phase trials assessing agents targeting this pathway, indicates that combination regimens with other classes of antitumor agents with a sufficiently wide therapeutic window, will be necessary.
The enzyme argininosuccinate synthetase 1 (ASS1) leads to arginine biosynthesis from citrulline and is epigenetically suppressed in a high proportion of mesothelioma cell lines. Loss of ASS1 renders mesothelioma cells addicted to exogenous arginine, and this defect may be therapeutically exploited by pegylated arginine deiminase (ADI-PEG20), which works by clearing circulating arginine. Nonepithelioid (biphasic and sarcomatoid) MPM subtypes are characterized by a 75% rate of ASS1 loss and disease control rate (DCR) of this subgroup resulted 94% in the TRAP Phase I trialof ADI-PEG 20 combined with 1st-line pemetrexed and cisplatin chemotherapy. Results from the randomized, placebo-controlled, double-blind phase 2/3 global ATOMIC-meso trial (NCT02709512) in non-epithelioid MPM are awaited.
In conclusion, despite our improved understanding of the biology of MPM, response to targeted therapies is hampered by intra-tumor heterogeneity and it is still unclear whether most of the actionable mutations constitute clonal or sub-clonal driver events. Longitudinal prospective studies, such as the TRACERx study in lung cancer, aiming at elucidating mechanism of resistance to treatment, are still missing in MPM. Properly designed clinical trials, which stratify patients for predictive biomarkers, are warranted. To this regard, patients enrolled in the MiST trial (NCT03654833) are currently offered a specific study treatment (either the parp-inhibitor rucaparib, the CDK4/6 inhibitor abemaciclib, the combination of the PD-1 inhibitor pembrolizumab and the AXL inhibitor bemcentinib or the combination of the PD-L1 inhibitor atezolizumab and the antiangiogenic agent bevacizumab) determined by the results of the molecular panel testing of their diagnostic tumor block. The ones who exhibit positive testing in more than one biomarker, will potentially be eligible for a subsequent protocol upon disease progression. This trial design is aimed at providing a more tailored approach for MPM patients.
## Mesothelin targeted therapies
Mesothelin (MSLN) is a glycoprotein with high expression in epithelioid mesothelioma and low expression in normal tissues, thereby it represents an attractive target for several therapies. A phase II trial comparing amatuximab (an anti-MSLN chimeric monoclonal antibody) plus first-line chemotherapy vs. chemotherapy alone was prematurely stopped in January 2017, not because of unacceptable toxicity but because of business reasons (NCT02357147).
According to a public announcement, anetumab ravtansine (an antibody-drug conjugate made by combining a human anti-MSLN antibody and the maytansinoid tubulin inhibitor DM4) also failed to improve PFS compared to vinorelbine in a randomized phase II trial for patients progressing after first-line (NCT02610140).
CRS-207 is a live, attenuated, non-virulent, Listeria monocytogenes (LADD) encoding human MSLN. After receiving two priming infusions of CRS-207, followed by pemetrexed/cisplatin chemotherapy, and CRS-207 booster infusions in a phase Ib trial, 89% (31/35) of patients had disease control; one complete response (3%) and 19 partial responses (54%) were reported. Reduction of tumor size was also observed post-CRS-207 infusion prior to chemotherapy in 11 patients and no treatment-related serious adverse events or deaths were observed. These results suggested that combining CRS-207 with traditional chemotherapy might potentially result in increased anti-tumor activity. However, after a phase II trial had showed no clinical activity of the combination of CRS-207 with PD-1 inhibition (NCT03175172), clinical development of this therapy was discontinued.
LMB-100 is a next generation immunotoxin against MSLN that consists of a humanized fragment of the anti-MSLN Fab bound to a de-immunized Pseudomonas exotoxin (PE). This PE-fusion protein has been engineered to decrease its immunogenicity. A Phase I, open-label study to investigate the safety, pharmacokinetics, and activity of LMB-100 in relapsed MPM patients is planned to complete accrual this year (NCT02798536).
Evaluating new combinations of MSLN directed therapies with checkpoint inhibitors and integrating MSLN targeting into new approaches such as adoptive T cell transfer might constitute the next step in the field, as first results have been promising.
## Immunotherapy
## Immune checkpoint inhibitors
The immune system is known to play a key role in MPM. Immune suppression locally induced by the tumor is high. Survival of patients with MPM is longer when tumors are highly infiltrated by cytotoxic CD8 + T cells (tumor-infiltrating lymphocytes), whereas PD-L1 expression is associated with shorter survival (median OS 5.0 in patients who are PDL1positive vs. 14·5 months PDL1-negative patients; p < 0.0001). Due to their ability to restore the capacity of immune system to counterattack tumor growth, CIs (directed toward CTLA4, PD1, PDL1 or their combinations) started to be investigated in MPM patients. A large randomized phase IIb trial, assessing tremelimumab, an anti-CTLA4 mAb, vs. placebo in a second or third-line setting did not show superiority of the immunotherapy in terms of OS. Looking at agents targeting the PD-1/PD-L1 pathway, interesting results were reported in the first early phase trials with overall response rates (ORR) ranging from 9 to 29% in patients previously treated with chemotherapy.
As shown in other types of cancer, combining CTLA-4 and PD-(L)1 mAb might further improve outcomes. In a single-center, single-arm, phase II trial (INITIATE) (86), the combination of ipilimumab and nivolumab for the treatment of recurrent MPM was assessed. Of the 34 patients evaluated for radiological response at 12 weeks, ten (29%) patients were partial responder and 13 (38%) had stable disease; adverse events were quite frequent (94% of patients) with 12 (34%) patients reporting grade 3 toxicity. Another randomized, non-comparative, openlabel, phase 2 trial (MAPS2), conducted in 21 hospitals in France (87), met its primary endpoint of DCR after randomization in the first 108 patients. This trial aimed to assess the anti-PD1 mAb alone (nivolumab) or in combination with anti-CTLA4 (ipilimumab) mAb in MPM patients who progressed to firstline chemotherapy. Twenty-four (DCR 44%) of 54 patients treated with nivolumab and 27 (DCR 50%) of 54 patients treated with nivolumab plus ipilimumab achieved disease control at 12 weeks. Objective responses were ten (19%) with nivolumab and 15 (28%) with nivolumab plus ipilimumab. Again, the safety profile was consistent with previous data on the combination. To note, three (5%) treatment-related death were reported with the combination (one fulminant hepatitis, one encephalitis, and one acute kidney failure).
These findings confirm the promising activity of both single and double check-point blockade in MPM patients who have relapsed. However, data presented at 2019 ESMO conference from the European Thoracic Oncology Platform (ETOP 9-15) PROMISE-meso randomized phase III trial (NCT02991482) comparing PD-1 inhibition with pembrolizumab to institutional choice single agent CT (gemcitabine or vinorelbine) as secondline treatment failed to show superiority of PD-1 treatment. Nearly four times more patients responded to immunotherapy (ORRs were 22% with pembrolizumab vs. 6% in CT, p = 0.004), but these responses were not translated into delayed progression or improved survival (median PFS was 2.5 months (95% CI 2.1-4.2) with pembrolizumab and 3.4 months (95% CI 2.2-4.3) with chemotherapy, HR 1.06 (95% CI 0.73-1.53), p = 0.76). In this study long-term responders to pembrolizumab were also found, again underlining the importance of understanding which patients should receive this treatment instead of chemotherapy. Data from another randomized trial comparing nivolumab vs. placebo in patients pre-treated with at least two lines of chemotherapy [NCT03063450 (CONFIRM)], are also warranted in order to select the best strategy. At the current time, results from the MAPS2 trial supported the National Comprehensive Cancer Network (NCCN) panel decision to introduce either nivolumab or nivolumab plus ipilimumab as treatment options in relapsed MPM patients and nivolumab was approved in Japan as second-line treatment after results from a multicenter, openlabel, single-arm, Japanese phase II study in MPM (MERIT) were reported, with ten (29%) patients showing an objective response.
Similar to other cancers, there might be a subgroup of MPM patients who might obtain a larger benefit from CIs, but relevant biomarkers have not been determined yet. Tumor PD-L1 IHC expression (with a cut-off of 1%) was correlated to ORR in both groups of MAPS-2 trial (nivolumab alone or nivolumab combined with ipilimumab) (87) but resulted in a better OS only in the nivolumab group. These correlations were not consistent in another phase II trial with nivolumaband, although PD-L1 status may be associated with sensitivity to CIs, also patients with low PD-L1 expression benefit from this treatment, with a reported ORR of 11.1%. Intra-patient heterogeneity, different cut-points for PD-L1 positivity and lack of assay standardization also prevent PD-L1 from being used as the only selection criteria for CIs-treatment in MPM. This should lead researchers to investigate other tumor and patients' characteristics (histological subtype, performance status, bloodderived tests) to get an upfront identification of patients who are likely to respond to CIs and integration of multiple parameters (infiltration of CD8 and other subpopulations of T-cells, genomic signatures, specific mutations, expression of different checkpoint inhibitors) beyond PD-L1 status will be crucial.
To improve response rate to CIs in MPM patients, two options may be pursued. The first one is to move CIs toward the first-line setting, where the reinvigoration of the immune system may be stronger and more efficient, and to combine them with chemotherapy, similar to what happened in non-small cell lung cancer. Results of the addition of the PD-L1 inhibitor durvalumab to cisplatin and pemetrexed were presented in form of an abstract at the 2018 World Conference on Lung Cancer, showing a PFS of 6.2 months with a 48% ORR in the context of a non-randomized phase II trial-ORR is 41.3% with first-line chemotherapy alone, as historically reported. In the United States, a similar phase II trial investigating durvalumab (MEDI4736) in combination with chemotherapy for first-line treatment of MPM is currently in the analysis phase (NCT02899195). The addition of either pembrolizumab (NCT02784171) or nivolumab (in a Japanese population)to chemotherapy is also being studied. The combination of ipilimumab and nivolumab is being compared with the cytotoxic chemotherapy standard in the first-line setting as well, with about 600 patients expected to be enrolled in a phase III trial.
The second option may be to combine CIs with either different immune-modulatory molecules, targeted therapies, antiangiogenic agents, or radiotherapy. Additional co-inhibitory and co-stimulatory molecules such as T-cell immunoglobulin and mucin-domain containing-3 (TIM3, also known as HAVCR2), lymphocyte activation gene 3 (LAG3) and inducible T cell co-stimulator (ICOS) are being investigated in mesothelioma. Inhibiting FAK together with PD-1, may enhance immune cell-associated antitumor cytotoxicity in vivo, which is hampered by expression of PD-L1and this represented the rationale for a phase I/IIa currently ongoing (NCT02758587). Similarly, in addition to the direct anti-tumor effects, pegylated arginine deiminase (ADI-PEG 20) may boost tumor immune surveillance and might be a good primer for an additional anti-tumor immune therapy, raising the question whether combining ADI-PEG 20 with PD-1/PD-L1 blockers may further enhance these drugs' anti-tumor efficacy.
Early phase trials also assessed the combination of anti-PD1/PDL1 agents and MSLN-directed therapies (in MSLNpositive patients). After results from a pre-clinical murine lung tumor model (CT26hMeso) demonstrated anti-PD1 enhanced LADD-induced tumor response (102), a phase 2 single-arm study of CRS-207 with pembrolizumab in relapsed MPM was started but no responses were showed, and the study was discontinued. Two other phase 2 trials (NCT03644550, NCT03126630) assessing the combination of pembrolizumab with the anti-MSLN Immunotoxin LMB-100 and with the antibody-drug conjugate anetumab ravtansine are currently enrolling patients, with the latter one also randomizing patients to pembrolizumab alone as active comparator.
Growing evidence that pro-angiogenesis factors have immunosuppressive activity has led researchers to evaluate the potentially synergistic combination of antiangiogenic agents and immunotherapy also in the treatment of MPM. VEGF signaling has been shown to attenuate the immune antitumor response by either influencing lymphocyte trafficking across endothelia to the tumor or directly inducing inhibitory immune cell subsets. Several trials are aiming to address whether the combination of CIs and antiangiogenic agents (either mAbs as bevacizumab and ramcirumab or TKIs as nintedanib) is able to improve outcomes in MPM patients (NCT03762018, NCT02856425, NCT03502746).
Finally, similarly to certain types of chemotherapy, radiotherapy can be exploited for its ability to cause immunogenic cell death (ICD), thus priming the release of damage-associated molecular patterns (DAMPs) and tumor-associated antigens (TAAs) and inducing a systemic anti-tumor immune response, that may be further enhanced by PD-1 (pembrolizumab) or PD-L1 (avelumab) blockade (NCT02959463, NCT03399552).
## Vaccines
Vaccines represent another way to boost the immune system activation against the tumor. Both protein, vector and cell-based vaccines have been tested in MPM.
Galinpepimut-S is a WT-1 synthetic peptide vaccine made out of molecules similar to those in the WT1 protein. After a phase II trial confirmed vaccine's safety when administered in the adjuvant setting, researchers' efforts are currently directed toward the assessment of the combination of galinpepimut-S and nivolumab (NCT04040231). It has been hypothesized that the negative influence of tumor microenvironment factors on the immune response might be mitigated by nivolumab, thus providing the opportunity for the reinvigorated immune cells, specifically sensitized against WT1 by the vaccine, to invade and destroy cancerous growth deposits.
Dendritic cells are antigen-presenting cells that present tumor-associated antigens (TAAs) to the immune system by trafficking from tumors to lymph nodes. They are essential in priming proliferation and activation of CD8 + cytotoxic Tlymphocytes and CD4 + helper T-lymphocytes resulting in a potent and specific anti-tumor response. Dendritic cell function is hampered in cancer patients by tumor-derived soluble factors that suppress their immune-stimulatory ability. However, dendritic cells can be generated in large amounts ex vivo and loaded with TAAs, prompting their recent usage as cancer vaccines in several neoplasms, including MPM. Several sources of tumor antigens (mRNA, peptides, proteins or whole tumor cell lysate) can be used to load DCs. Because TAAs are difficult to identify in mesothelioma (thus excluding peptides as best source), and adequate tumor tissue is rarely obtained from mesothelioma patients, an allogenic tumor lysate has been developed. Results from a first-inhuman clinical trial involving nine MPM (non-progressive after at least 4 cycles of chemotherapy) showed that this approach is safe (no dose-limiting toxicities were established) and led to radiological responses and promising survival data, with median PFS of 8.8 months and median OS not reached. A large multicentric phase II/III randomized trial with allogeneic-lysate pulsed dendritic cell immunotherapy as maintenance treatment after platinum-based chemotherapy is currently enrolling in Europe [NCT03610360 (DENIM)] (111).
## T cell therapies
Another promising cell-based strategy in mesothelioma is represented by adoptive T cell therapy. Data from a phase I trial investigating chimeric antigen receptor (CAR) T cell therapy targeted to the MSLN protein in 19 MPM patients progressed following standard platinum-based chemotherapy were recently reported. A single-dose of second-generation CD28costimulated MSLN-CAR T cells with the Icaspase-9 safety gene (IcasM28z) was given intrapleurally (as recommended by previous observations in murine models, in which intrapleural administration vastly outperformed intravenous infusion)with or without cyclophosphamide preconditioning. No evidence of on-target, off-tumor or therapy related toxicity was seen, and CAR T-cell persistence was associated with decreased levels of serum soluble MSLN-related peptide (SMRP) levels (>50% compared to pretreatment) and evidence of tumor response. Of the 14 patients who received anti-PD1 agents, off-protocol, after the CAR T-cell therapy, 2 achieved a complete metabolic response, 5 obtained a partial response, and 4 had stable disease. Combining anti-PD1 therapy with CAR T cells is also supported by prior preclinical data showing that CAR T cells become functionally exhausted in the presence of a large tumor burden and that anti-PD-1 therapy can reactivate these exhausted cells.
## Virotherapy
Oncolytic viral therapy represented in the last decades an emerging field of immunotherapy and a promising experimental strategy. Viruses can act by infecting cancer cells and leading to cell lysis after replication. This renders tumor-associated and viral antigens recognizable to the immune system, thus triggering antitumor immune responses (viroimmunotherapy). Oncolytic viruses need also to be tumor selective, and although malignant cell-specific oncolysis naturally occurs because of the impairment of the type I interferon pathway in many tumor cells, viruses may be engineered in order to increase their selectivity. Viruses may be used also for gene therapy, thereby therapeutically changing the infected tumor cells by gene transfer.
The pleural location and the peculiar pattern of growth (mostly localized), which provide access to direct intratumoral injection of virus, make MPM an ideal candidate for assessing the efficacy of oncolysis. The safety of virotherapy has been assessed and some clinical response have been reported. Among the many viral vectors that have been investigated, the recombinant replication incompetent adenoviral (ADV) vector encoding human interferon-α (IFNα, a naturally-occurring protein with anti-cancer properties) administered "in situ" (intrapleurally) with celecoxib (to reduce the number of immunosuppressive MDSCs) before chemotherapy, was well tolerated and appeared to improve overall survival rates. Combinations of virotherapy with CIs, chemotherapy, and radiation are expected to further boost the effects on antitumor immunity and represent the object of ongoing trials, such as the phase III INFINITE trial (NCT03710876), in which about 300 patients will receive gemcitabine and celecoxib with or without the ADV-delivered IFNα-2b (rAd-IFN).
# Conclusion
In the past two decades there was limited success in the development of novel therapies for MPM. Multiple biases in the design of clinical trials and the peculiar biological features of MPM were most probably responsible for delaying the discovery of effective therapeutic agents. Most of the previous trials attempted to readapt drugs that succeeded in other cancer types to MPM. However, they were either too small or not stratified for predictive biomarkers. Results from phase II studies were often not replicated in larger, randomized, phase III trials, pointing out that well controlled trials with appropriate size and duration are crucial to confirm the efficacy of a new agent.
In the last few years, mesothelioma genetics, epigenetics, and the tumor microenvironment (especially immune-biology) have been studied more deeply and this knowledge has started to be properly applied to discover new therapies. In particular, expectations are now high that CIs and other immunotherapies will have a leading role in the future therapeutic armamentarium of MPM. Noteworthy, scientific evidence supporting the use of CIs in MPM are still incomplete, mainly based on nonrandomized studies with surrogate end-points and they have not been always replicated in the real-life context. Because of the risk of cumulative toxicities and of the high cost of these drugs (especially of combinations), validated biomarkers are urgently needed to select MPM patients who may benefit from immunotherapies. Since the "one-size fits all" approach is not recommended for immunotherapy and MPM and the efficacy of CIs is still to be established in a larger population, there is still a need for new treatments in MPM and the implementation of other targeted agents is eagerly awaited.
Only a close collaboration between medical centers and industry may lead to the conduction of well-designed, biomarker-driven clinical trials. New trials should always include translational and quality of life components, in order to clarify the molecular basis of response or progression to treatments and to finally improve the degree of reliability of the possible benefit of new therapies for MPM. |
Use of Muller's maneuver in the evaluation of patients with sleep apnea - literature review
Sl eep apnea-hypopnea syndrome was described twenty years ago, and since then there have been doubts and controversies regarding it. Fiberoptic nasopharyngoscopy with Muller's maneuver, first described by Borowieck and Sassin (1983), is among them. Aim: Careful literature review on Muller's maneuver, regarding whether it can predict the sucess of uvulopalatopharyngoplasty, location of upper airway obstruction and severity of the disorder. Discussion and literature rewiew: Literature has shown that there isn't a consensus about the use of Muller's maneuver. In spite of being technically easy, inexpensive and widely used, it is very unespecific and subjective. Conclusion: The importance of Muller's maneuver in evaluating apneic patients has been questioned, because there are controversies whether it can predict the sucess of uvulopalatopharyngoplasty, location of upper airway obstruction and severity of the disease.
# Introduction
Studies involving the main characteristics of patients with Obstructive Sleep Apnea Syndrome (OSAS) started with Burwell, Robin, Waley and Bickelmann (1956) [bib_ref] Extreme obesity associated with alveolar hypoventilation: a Pickwickian syndrome, Burwell [/bib_ref]. They described the Pickwickian Syndrome, in homage to the English writer Charles Dickens, author of the classic "The Posthumous Papers of the Pickwickian Club" (1837), which main character was an obese, sleepy and snoring boy. The syndrome is classically made up of obesity, hypercapnia, cor pulmonale, erithrocytosis and excessive day-time sleepiness.
Obesity causes overload and consequent respiratory depression, leading to hypercapnia and hypoxemia; such blood gas unbalance could explain day-time excessive sleepiness [bib_ref] Clinical and physiological aspects of a case of obesity, polycythemia and alveolar..., Auchinloss [/bib_ref] [bib_ref] Neurophysiological studies of abnormal night sleep and Pickwickian syndrome, Jung [/bib_ref].
During the 60's, when polysomnography (PSG) became available, European authors started to investigate the Pickwickian syndrome as a sleep respiratory disorder and concluded that day-time excessive sleepiness came from sleep fragmentation, and not from changes in blood gases [bib_ref] Neurophysiological studies of abnormal night sleep and Pickwickian syndrome, Jung [/bib_ref] [bib_ref] Polygrafic study of episodic diurnal and nocturnal (hypnic and respiratory) manifestations of..., Gastaut [/bib_ref].
In Italy, 1972, the first sleep-related symposium on respiratory disorders was held, when Guilleminault et al. [bib_ref] The sleep apnea syndromes, Guilleminault [/bib_ref] established the terminology: Obstructive Sleep Apnea Syndrome (OSAS), characterized by excessive day-time sleepiness and apnea episodes detected by PSG. The concept of hypopnea was first described in 1979 as superficial breathing causing denaturation during sleep [bib_ref] Sleep apnea, hypopnea and oxygen desaturation in normal subjects, Block [/bib_ref]. It was almost a decade later, in 1988, that the term OSA was established [bib_ref] The sleep hypopnea syndrome, Gould [/bib_ref].
According to the American Academy of Sleep Disorders, in 2005 8 , the proper terminology is OSAS, since there is no evidence that the physiopathology and the clinical repercussions of the apnea and hypopnea events are different.
Numerous theories have been proposed to explain the disease pathophysiology, which is multifactorial, partially stemming from anatomical variations of the upper airways and the facial skeleton associated with neuromuscular alterations in the pharynx [bib_ref] Pathophysiology of upper airway obstruction during sleep, Badr [/bib_ref] [bib_ref] Anatomy and Physiology of Upper Airway Obstruction, Kuna [/bib_ref]. Despite our knowledge that apneic individuals have pharynxes more prone to colapse 11-13 , we still have a great difficulty in assessing exactly which point in the pharynx suffers the worst collapse during sleep. That happens because the evaluations are all carried out with the patient awake, when his/her muscle tone is maintained and during sleep there is a progressive hypotonia, reaching full atony during the REM ("rapid eye movement") sleep.
Since the upper airway (UAW) is the OSA obstruction site and the working ground of otorhinolaryngologists, it has been carefully studied by specialists of the field. In these regards, the Nasofibroscopy-Assisted Muller's Maneuver (NMM) through Nasofibroscopy, described in 1983, is the scope of the present paper.
## Goal
Literature review, with a critical and comparative analysis regarding the NMM's capacity to detect the upper airway point of collapse, to predict the success of uvulopalatopharyngoplasty (UPPP) and the OSA severity.
# Method
Asystematic review. We studied papers indexed in the LILACS and MEDLINE databases from 1950 all the way to 2007.
## Literature review and discussion
In 1964 the first study was published involving oropharynx characteristics in snoring adults, showed that 91% of these patients had a narrow pharynx, elongated soft palate and uvula, in which they proposed the mucosal resection of the anterior tonsillar pillar and part of the uvula, with satisfactory results.
These ideas were refined at the same time that studies started to confirm the hypothesis that the oropharynx was an important OAS obstruction site. Based on these facts, in 1981 17 the UPPP was described, with a 50% success rate, a true landmark in the treatment of this disorder. At this time, the UPPP became quite popular, being the most frequently performed procedure for the treatment of snoring and sleep apnea during the 80's and 90's 18 . Since then, otolaryngologists took on a very important role in OSA diagnosis and treatment.
Nonetheless, surgical results were better in patients with snoring and only 50% of the apneic patients benefited from the surgery -50% of HAI (hypopnea Apnea Index) reduction was considered a success [bib_ref] Snorig, and some obstructive sleep apnea, can be cured by oropharyngeal surgery, Simmons [/bib_ref].
In later studies, when the success rate of 50% reduction in HAI was used, associated with a HAI below 20 , the success rate found was 40% 20 . Numerous factors were held responsible for these poor results, such as disease severity, multiple obstruction sites, obesity and anatomical alterations on the mandible and maxilla [bib_ref] The efficacy of surgical modifications of the upper airway in adults with..., Sher [/bib_ref]. The success of UPPP varies between 8 and 100% in the literature; however, there are numerous techniques and success criteria used, which make it very difficult to compare the data [bib_ref] Clinical staging for sleep disordered breathing, Friedman [/bib_ref] [bib_ref] Long-term results in the treatment of obstructive sleep apnea, Pirsig [/bib_ref] [bib_ref] Staging of Obstructive Sleep Apnea/ Hypopnea Syndrome: A Guide to Apropriate Treatment, Friedman [/bib_ref] [bib_ref] Does Severity of Obstructive Sleep Apnea/Hypopnea Syndrome Predict Uvulopalaopharyngoplasty?, Friedman [/bib_ref].
Even then, UPPP remains as the most frequently performed surgical procedure for the treatment of OAS [bib_ref] The efficacy of surgical modifications of the upper airway in adults with..., Sher [/bib_ref] [bib_ref] Clinical staging for sleep disordered breathing, Friedman [/bib_ref] [bib_ref] Staging of Obstructive Sleep Apnea/ Hypopnea Syndrome: A Guide to Appropriate Treatment, Friedman [/bib_ref] [bib_ref] Does Severity of Obstructive Sleep Apnea/Hypopnea Syndrome Predict Uvulopalaopharyngoplasty?, Friedman [/bib_ref].
With the intent of properly selecting the apneic patients that could be successfully treated by UPPP, determining a trend towards pharyngeal collapse and laxity, in 1983 14 the NMM was described, in which the patient undergoes a forced inhaling effort, having both the nose and mouth closed and the examiner, with the nasofibroscope located in the retrolingual region, observes the side-to-side and antero-posterior narrowing of the pharyngeal walls; the maneuver is repeated with the device in the retropalatal region. The patients with retropalatal collapse would be the best candidates to UPPP [bib_ref] The efficacy of surgical modification of upper airway in adults with obstructive..., Sher [/bib_ref].
The capacity of NMM in forecasting UPPP success was assessed in a group of apneic patients 31 , for which 30 patients with retropalatal collapse were studied through the NMM; of those, 22 (73%) had a minimum reduction of 50% in the hypopnea apnea index (HAI) in relation to baseline, considered a surgical success by the author; notwithstanding, in the literature the most accepted criterion is a minimum reduction of 50% in the HAI and HAI< 2018. The study was carried out in apneic patients only and surgery was offered only to those patients with retropalatal obstruction; thus the lack of a control group, as well as the success criterion used, limits a proper analysis of the results presented.
The same study was later on reproduced 32 in 24 patients with moderate or severe OSA, and surgery was performed in patients with retropalatal (15) and retrolingual (9) collapse. Success rate, using the same criteria aforementioned, in the first group was 33.3% [bib_ref] The sleep apnea syndromes, Guilleminault [/bib_ref] and that of failure in the second group was 77.7% (7). This study suggests that the NMM is not capable of predicting those that would have a good surgical result; however, it can select those that would fail.
A retrospective study was carried out in 30 patients with OSAS 33 and the NMM predictive value together with that of cephalometry were compared in the selection of patients for UPPP. NMM was not able to forecast those patients that would be successfully treated by UPPP (50% reduction in HAI and HAI< 20) or surgical failure. The authors propose a model grouping up three cephalometric measures (distance between the hyoid and the mandibular plane, skull-neck angle and length of the maxilla) and hypersomnia, which is able to properly select these patients in 83% of the cases.
A similar study to the one aforementioned, involving 53 apneic patients 34 considered an HAI reduction of 50% as surgical success. Both NMM and cephalometry were capable of selecting successful or unsuccessful patients for UPPP. The maneuver was carried out with the patient laying down and seating up, and there was no difference between them.
The studies aforementioned show that NMM alone is not a good method to detect the pharynx collapse site and predict UPPP success, since patients with retropalatal narrowing seen at NMM had surgical results short of what was expected.
In 1999, Ritter et al. [bib_ref] Quantitative evaluation of the upper airway during nasopharyngoscopy with the muller maneuver, Ritter [/bib_ref] published the only study in the literature with a quantitative analysis of NMM, using software to measure the area and the diameters of the retropalatal and retroglossal areas in the baseline and during the maneuver, and also a pressure transducer to control the patient's inspiratory effort. Thus, the authors excluded a major criticism to this procedure: patient and examiner's subjectiveness. The test was carried out in normal patients, laying down and in a seating position. In the retropalatal region, there was a reduction in the area and the side-to-side diameter during Muller's maneuver when compared to baseline values. The retrolingual region did not show any significant area reduction, because besides the side-to-side narrowing, there was an antero-posterior diameter enlargement. In both, there was no difference between NMM laying down or seating up.
In a OSAS consensus held in 2002, Rombaux et al.suggest that NMM should be performed with a pressure transducer under a -20 cm H 2 O inspiratory effort to minimize patient`s subjectiveness to the test; however, they say it would not be feasible in daily practice. Nonetheless, its indication is maintained in the consensus because it is easy to perform, and it is time and cost effective.
Many patients are incapable of producing the proper inspiratory effort, and there may be differences among the patients and even different results from the same patient in different tests. Any test based on the patient's collaboration has some level of variability [bib_ref] Clinical standing for sleep-disordered breathing, Friedman [/bib_ref].
The maneuver's reproducibility was studied38 between two different examiners, one of them a first year resident and the other was a professor in the institution and there was a good correlation between them. Nonetheless, it is a renowned institution (Stanford University) to treat sleep disorders, with a large number of apneic patients and physicians used to and trained to assess them [bib_ref] Reliability of the Muller maneuver and its association with sleep-disordered breathing, Terris [/bib_ref].
By the same token, the agreement between the two examiners during NMM was also assessed 39 . Insofar as the pharyngeal narrowing is concerned, there was an agreement in 27 of 42 tests and disagreement in 15. In the retropalatal region, there was a good agreement (16 en 21 tests), with kappa index = 0.63. The results showed a weak agreement in the retroglossal region (11 in 21 tests), with a kappa index = 0.3.
Therefore, there is a tendency in the literature of stating that the NMM reproducibility by examiners is challengeable.
Studying the correlation with OSAS severity, one can conclude that the Muller's maneuver is not a good disease severity predictor40. Other authors found a moderate agreement (72%) [bib_ref] Reliability of the Muller maneuver and its association with sleep-disordered breathing, Terris [/bib_ref].
According to the literature, NMM advantages are: easy to do for patient and examiner, time and cost effective and broadly divulged [bib_ref] Available techniques for objective assessment of upper airway narrowing in snoring and..., Faber [/bib_ref]. The disadvantaged are: invasive, subjective for the patient and examiner, questionable reproducibility, possible dynamic modification of the upper airway dynamics because of the endoscope present, which does not necessarily reproduces the apnea episode [bib_ref] The predictive efficacy of the muller maneuver in uvulopalatopharyngoplasty, Katsantonis [/bib_ref] [bib_ref] Clinical standing for sleep-disordered breathing, Friedman [/bib_ref] [bib_ref] Available techniques for objective assessment of upper airway narrowing in snoring and..., Faber [/bib_ref].
## Final remarks
Muller's maneuver is a tool used to help the otolaryngologist in handling the apneic patient; however its relevance in testing has been challenged, since there are controversies in the literature as to its capacity to detect the upper airway collapse site, of predicting the oropharyngeal surgery success and OSAS severity.
In our service, we used the nasofibroscopy to complement the neck and orofacial physical exam in patients with suspected OSAS; however the Muller's maneuver has a fundamentally didactic intent, with little input regarding the treatment approach for the apneic patient.
BRAZILIAN JOURNAL OF OTORHINOLARYNGOLOGY 75 (3) MAY/JUNE 2009 http://www.rborl.org.br / e-mail: [email protected] |
Graphene: The Missing Piece for Cancer Diagnosis?
This paper reviews recent advances in graphene-based biosensors development in order to obtain smaller and more portable devices with better performance for earlier cancer detection. In fact, the potential of Graphene for sensitive detection and chemical/biological free-label applications results from its exceptional physicochemical properties such as high electrical and thermal conductivity, aspect-ratio, optical transparency and remarkable mechanical and chemical stability. Herein we start by providing a general overview of the types of graphene and its derivatives, briefly describing the synthesis procedure and main properties. It follows the reference to different routes to engineer the graphene surface for sensing applications with organic biomolecules and nanoparticles for the development of advanced biosensing platforms able to detect/quantify the characteristic cancer biomolecules in biological fluids or overexpressed on cancerous cells surface with elevated sensitivity, selectivity and stability. We then describe the application of graphene in optical imaging methods such as photoluminescence and Raman imaging, electrochemical sensors for enzymatic biosensing, DNA sensing, and immunosensing. The bioquantification of cancer biomarkers and cells is finally discussed, particularly electrochemical methods such as voltammetry and amperometry which are generally adopted transducing techniques for the development of graphene based sensors for biosensing due to their simplicity, high sensitivity and low-cost. To close, we discuss the major challenges that graphene based biosensors must overcome in order to reach the necessary standards for the early detection of cancer biomarkers by providing reliable information about the patient disease stage.
# Introduction
Cancer is considered to be one of the leading causes of health loss worldwide and both its early diagnosis and efficient treatment still present challenges to overcome, despite great progress made in the past few decades [bib_ref] Diagnosis and management of lymphomas and other cancers in hiv-infected patients, Carbone [/bib_ref] [bib_ref] Diagnosis and management of intradiverticular bladder tumours, Walker [/bib_ref]. The current standard controlling of cancer patients involves stage determination (by imaging or biopsies), chemo/radiation therapy, and/or surgical resection. Different molecular imaging techniques, including optical imaging by fluorescence and Raman spectroscopy have furthered our understanding of cancer initiation, progression, and metastasis [bib_ref] Theranostic applications of carbon nanomaterials in cancer: Focus on imaging and cargo..., Chen [/bib_ref]. Molecular imaging is also a useful tool to monitor in vivo biochemical events that can provide information about cancer diagnosis and therapeutic response [bib_ref] A molecular imaging primer: Modalities, imaging agents, and applications, James [/bib_ref].
The aspired goal in an accurate and early-stage diagnosis of cancer is based on the detection and quantification of reliable cancer biomarkers by cost-effective and less invasive methods. Researchers believe that early diagnosis of diseases with minimal cost and time-consumption may become achievable due to recent advances in the development of biosensors. These devices use biorecognition elements for the selective interaction with an analyte and different types of transducers to obtain the signal readout. The operational characteristics of biosensors have been reported as improving substantially when nanomaterials such as graphene and its derivates are employed. Graphene is aromatic, hydrophobic and chemical inert; moreover, it is biocompatible and has the facility to adsorb biomolecules due to π-π stacking between its hexagonal cells and the carbon rings present in the majority of nano/biomolecules. Owing to its exceptional charge mobility and atomic thickness, graphene have been proposed as the basis to sensitive detection and chemical/biological free-label [bib_ref] Graphene-like two-dimensional layered nanomaterials: Applications in biosensors and nanomedicine, Yang [/bib_ref] [bib_ref] Carbon nanostructure-based field-effect transistors for label-free chemical/biological sensors, Hu [/bib_ref]. Furthermore, since graphene is a two-dimension nanostructure, it does not present the geometric constrains observed for other carbon-based devices. Additionally, graphene oxide (GO), the oxidative form of graphene has attracted the attention of scientists because its surface chemistry is highly versatile due to the presence of oxygen groups capable of enabling not only the use of several functionalization approaches but also the preparation of multifunctional nanocomposites, extending the range of the applications of these nanomaterials in biosensores.
Herein we intent to discuss the most relevant graphene based technology for the development of different types of biosensors skillful to detect characteristic cancer biomolecules in early stages or overexpressed on cancerous cells surface. We summarize the current existing challenges on the cancer biomarker detection and quantification through graphene based biosensors in order to increase the selectivity and sensitivity. Finally, we intent to give a global view about the challenges for future development of various types of graphene based biosensors for cancer detection.
## Graphene and its derivatives
Theoretically, graphene sheets are perfect 2D single crystal formed by sp 2 -hybridized carbon bonds in aromatic structure. Although at the moment several synthetic methodologies allow to obtain this material, most of them are really far from provide the theoretical structure, producing instead graphene sheets with different chemical or/and physical defects. The most common defects observed on "graphene" are: sheets with different numbers of atomic layers that can go from monolayer until multilayer; the atomic flatness is not real because the sheets tend to distort due to the presence of structural defects such as lattice missing carbon atoms or inclusion of other foreign atoms, in particular oxygen; and different lateral dimensions of the sheets [bib_ref] Carbon nanomaterials in biosensors: Should you use nanotubes or graphene?, Yang [/bib_ref]. These imperfections have a strong influence on the final optical and electrical properties of graphene and in many cases are related with the synthesis method used for its fabrication [bib_ref] Synthesis, Characterization, and Selected Properties of Graphene, Rao [/bib_ref]. In order to clearly and consistently describe the various derivatives of graphene, graphene based materials can be classified according to three major physical-chemical features (number of layers, quantity of oxygen and lateral dimensions) [bib_ref] Classification framework for graphene-based materials, Wick [/bib_ref]. This standardization of graphene materials offers the possibility to have an effective control of the major factors that influence graphene properties, which is really important for the development of sensors in a reproducible and consistent way.
Hereafter we describe the family of graphene and its derivatives [fig_ref] Figure 1: Illustration of graphene derivatives with potential applications on cancer biosensors [/fig_ref] , their main properties and preparation methods as well as some methodologies used to surface modify these nanostructures and consequently increase their sensing performance. biorecognition elements for the selective interaction with an analyte and different types of transducers to obtain the signal readout. The operational characteristics of biosensors have been reported as improving substantially when nanomaterials such as graphene and its derivates are employed. Graphene is aromatic, hydrophobic and chemical inert; moreover, it is biocompatible and has the facility to adsorb biomolecules due to π-π stacking between its hexagonal cells and the carbon rings present in the majority of nano/biomolecules. Owing to its exceptional charge mobility and atomic thickness, graphene have been proposed as the basis to sensitive detection and chemical/biological free-label [bib_ref] Graphene-like two-dimensional layered nanomaterials: Applications in biosensors and nanomedicine, Yang [/bib_ref] [bib_ref] Carbon nanostructure-based field-effect transistors for label-free chemical/biological sensors, Hu [/bib_ref]. Furthermore, since graphene is a two-dimension nanostructure, it does not present the geometric constrains observed for other carbon-based devices. Additionally, graphene oxide (GO), the oxidative form of graphene has attracted the attention of scientists because its surface chemistry is highly versatile due to the presence of oxygen groups capable of enabling not only the use of several functionalization approaches but also the preparation of multifunctional nanocomposites, extending the range of the applications of these nanomaterials in biosensores.
Herein we intent to discuss the most relevant graphene based technology for the development of different types of biosensors skillful to detect characteristic cancer biomolecules in early stages or overexpressed on cancerous cells surface. We summarize the current existing challenges on the cancer biomarker detection and quantification through graphene based biosensors in order to increase the selectivity and sensitivity. Finally, we intent to give a global view about the challenges for future development of various types of graphene based biosensors for cancer detection.
## Graphene and its derivatives
Theoretically, graphene sheets are perfect 2D single crystal formed by sp 2 -hybridized carbon bonds in aromatic structure. Although at the moment several synthetic methodologies allow to obtain this material, most of them are really far from provide the theoretical structure, producing instead graphene sheets with different chemical or/and physical defects. The most common defects observed on "graphene" are: sheets with different numbers of atomic layers that can go from monolayer until multilayer; the atomic flatness is not real because the sheets tend to distort due to the presence of structural defects such as lattice missing carbon atoms or inclusion of other foreign atoms, in particular oxygen; and different lateral dimensions of the sheets [bib_ref] Carbon nanomaterials in biosensors: Should you use nanotubes or graphene?, Yang [/bib_ref]. These imperfections have a strong influence on the final optical and electrical properties of graphene and in many cases are related with the synthesis method used for its fabrication [bib_ref] Synthesis, Characterization, and Selected Properties of Graphene, Rao [/bib_ref]. In order to clearly and consistently describe the various derivatives of graphene, graphene based materials can be classified according to three major physical-chemical features (number of layers, quantity of oxygen and lateral dimensions) [bib_ref] Classification framework for graphene-based materials, Wick [/bib_ref]. This standardization of graphene materials offers the possibility to have an effective control of the major factors that influence graphene properties, which is really important for the development of sensors in a reproducible and consistent way.
Hereafter we describe the family of graphene and its derivatives [fig_ref] Figure 1: Illustration of graphene derivatives with potential applications on cancer biosensors [/fig_ref] , their main properties and preparation methods as well as some methodologies used to surface modify these nanostructures and consequently increase their sensing performance.
## Graphene
Chemical vapor deposition (CVD) production of graphene offers the possibility to obtain large sized films that are ideally for the fabrication of electronic sensor devices with high specific detection area and low noise [bib_ref] Low-frequency 1/f noise in graphene devices, Balandin [/bib_ref]. However, some problems can be found when using this type of methodology since graphene can present some defects and impurities and few-layered domains that really affects the electron mobility and limits the size of device fabrication. Some practical problems associated with the need to transfer the produced graphene sheets to insulating substrates for the sensor development are reported elsewhere [bib_ref] Chemical vapor deposition of graphene single crystals, Yan [/bib_ref]. Despite the limitations associated with the this synthesis method, recently a chemiresistive sensor based on a single layer graphene produced by CVD was reported to exhibit excellent selectivity toward eleven chemically distinct compounds and nine mono-substituted benzene compounds with an accuracy of 96% and 92%, respectively. The sensor used throughout each experiment did not have any surface modifications to enhance selectivity; and therefore, the observed responses were inherent to the material and device itself. These results are very a promising for future development of multiarrays graphene based sensor.
The synthesis of large graphene sheets is also possible through the epitaxial growth on the basal plane of single-crystal silicon carbide. The big advantage of this approach is that graphene films formed at silicon carbide (SiC) surface can be directly used for sensor development since the substrate is a good insulator and the strong interaction between graphene and SiC creates a graphene bandgap to 0.26 eV, which is ideal for sensors whose detection is based in induced field effect [bib_ref] Substrate-induced bandgap opening in epitaxial graphene, Zhou [/bib_ref]. However graphene growth at SiC surface shows some typical defects such as substrate induced corrugations and rotational disorder between layers resulting in films with heterogeneous thickness [bib_ref] Room-temperature molecular-resolution characterization of self-assembled organic monolayers on epitaxial graphene, Wang [/bib_ref].
## Graphene oxide
The wet chemical exfoliation of graphite is a very common adopted approach for the synthesis of GO in large scale and at low cost. GO is an electrical insulator, chemically heterogeneous with high amount of oxygen groups linked to the carbon lattice forming defect regions with sp 3 C-C bonds [bib_ref] Chemical methods for the production of graphenes, Park [/bib_ref]. GO has a huge potential for the development of optical sensors due to its particular properties for optical sensing: photoluminescence over a broad range of wavelengths and highly efficiency as a fluorescence quencher [bib_ref] Graphene oxide as a chemically tunable platform for optical applications, Loh [/bib_ref]. Furthermore, GO can also be chemically engineered in order the tune its optoelectronic properties, from insulator to semiconductor or semi-metal, by applying chemical, thermal or electrochemical reduction methods [bib_ref] Chemically derived graphene oxide: Towards large-area thin-film electronics and optoelectronics, Eda [/bib_ref]. The resultant material, reduced graphene oxide (rGO), is characterized to have high degree of defects on carbon sp 2 lattice and residual oxygen contents (dependent of the reducing method applied). Nevertheless, rGO is considered the most promising material for electrochemical sensors, since it is electrochemically more active than any other graphene family member [bib_ref] Graphene-based nanomaterials and their electrochemistry, Pumera [/bib_ref]. Furthermore, rGO has an interesting potential for the development of electronic sensing due to its capability to acquire high on-off ratio dependent of applied voltage [bib_ref] Insulator to semimetal transition in graphene oxide, Eda [/bib_ref]. The pioneer work on the development of electrical biosensors using rGO was made by Mohanty and Berry [bib_ref] Graphene-based single-bacterium resolution biodevice and dna transistor: Interfacing graphene derivatives with nanoscale..., Mohanty [/bib_ref]. They demonstrated the viability of using rGO as sensitive building block for bioelectronics at both microbial and molecular levels: as either a single bacterium resolution interfacial device, a label free reversible DNA detector or a polarity-specific molecular transistor for protein/DNA adsorption. After that many other works have been published using rGO for the development of electronic sensors [bib_ref] Electrochemistry of graphene and related materials, Ambrosi [/bib_ref].
## Nano-graphene and nano-graphene oxide
Graphene quantum dots (GQDs), also referred as Nano-graphene (NG) are nanometer-sized fragments with distinct properties from graphene, which makes them interesting candidates for a whole range of new applications. The methods to synthesize NG can be classified into two types: the top-down and bottom-up methods [bib_ref] Graphene quantum dots with controllable surface oxidation, tunable fluorescence and up-conversion emission, Zhu [/bib_ref] being the top-down routes that possess the advantages of having abundant raw materials, large scale production and simple operation the most applied. The top-down approaches include hydrothermal [bib_ref] Hydrothermal route for cutting graphene sheets into blue-luminescent graphene quantum dots, Pan [/bib_ref] , electrochemical [bib_ref] Facile synthesis of water-soluble, highly fluorescent graphene quantum dots as a robust..., Zhang [/bib_ref] , solvothermal and microwave-assisted cutting, between others. Recently, Li et al. [bib_ref] Focusing on luminescent graphene quantum dots: Current status and future perspectives, Li [/bib_ref] reported these methodologies in a systematic review. The bottom-up methodologies include mainly stepwise organic synthesis using organic molecules like polycyclic aromatic hydrocarbons [bib_ref] Bottom-up fabrication of photoluminescent graphene quantum dots with uniform morphology, Liu [/bib_ref] and also cage opening of fullerenes [bib_ref] Transforming c60 molecules into graphene quantum dots, Lu [/bib_ref]. Indeed, it is significant to synthesize stable colloidal NG with customized and controllable size in order to obtain tunable fluorescence NG [bib_ref] Synthesis of large, stable colloidal graphene quantum dots with tunable size, Yan [/bib_ref] which diameter distribution ranges from 3 nm to 20 nm [bib_ref] Graphene quantum dots: Emergent nanolights for bioimaging, sensors, catalysis and photovoltaic devices, Shen [/bib_ref].
Nano-graphene oxide (NGO) is a new class of carbon based materials that was proposed for biomedical applications due to its small size, intrinsic optical properties, large specific surface area, and easy to functionalize [bib_ref] Nano-graphene oxide: A potential multifunctional platform for cancer therapy, Gonçalves [/bib_ref]. The preparation methods for NGO are mainly based on top-down methodologies. The high degree of oxidation of the obtained GO grants this material with a high hydrophilic character which allows to obtain very stable aqueous colloidal suspensions and able for easily functionalization [bib_ref] Graphene oxide dispersions in organic solvents, Paredes [/bib_ref].
## Engineering graphene surface for sensing applications
One very simple way to induce further sensing capabilities to graphene is by changing its physical-chemical structure. The changes required to obtain new graphene features can be made by the chemical doping of its structure with different foreign atoms that promote changes in electrical, electrochemical and optical properties. The most typical methodologies for atomic modifications of graphene surface includes oxygenation, hydrogenation and fluorination by using different synthetic approaches [bib_ref] An introduction to the chemistry of graphene, Wang [/bib_ref]. Another very conventional way is by grafting graphene surface with recognition elements that can act as detection targets promoting specific interactions with an analyte. Many parameters should be considered during the engineering of the sensing surface of graphene: the specific sensing target, the sensing mechanism, performance (detection limit and dynamic range), reproducibility, cost and manufacturing.
## Graphene functionalization with small molecules and biostructures
There are two main different approaches for the surface functionalization of graphene based materials: covalent and noncovalent. The covalent functionalization is very straightforward when using as a precursor GO sheets. The presence of oxygen functional groups like carboxylic or hydroxyl at GO surface enables the establishment of covalent bonds with other molecules, polymers, biological structures and nanoparticles. The range of chemical mechanism available to design new covalent bonds between graphene and other organic entities is very broaden following the basic principles of organic chemistry [bib_ref] Harnessing the chemistry of graphene oxide, Dreyer [/bib_ref]. The organic covalent reactions of graphene include two general routes: the formation of covalent bonds between free radicals or dienophiles and sp 2 hybridized carbon bonds of graphene and/or the formation of covalent bonds (usually via amide or ester formation) between the functional molecule and the oxygen groups at graphene surface [bib_ref] Functionalization of graphene: Covalent and non-covalent approaches, derivatives and applications, Georgakilas [/bib_ref]. For example, oligothiophenes can be grafted on GO sheets through covalent amide bonds resulting in an improved absorption in the whole spectral region and an efficient quenching of photoluminescence able to exhibit enhanced nonlinear optical and optical limiting properties that can be explored for the development of new optoelectronic sensors [bib_ref] Synthesis, characterization and optical limiting property of covalently oligothiophene-functionalized graphene material, Liu [/bib_ref].
Many other examples are reported on the literature describe the covalent modification of GO at molecular level via charge transfer between electron donor and electron acceptor molecules allowing significant changes in the electronic structure of graphene [bib_ref] Donor-acceptor graphene-based hybrid materials facilitating photo-induced electron-transfer reactions, Stergiou [/bib_ref]. In general, covalent functionalization of GO leads to better performance than noncovalent functionalization due to a more effectively improved energy or electron transfer from the functional moiety to the graphene nanomaterial. Non-covalent bonds between GO and others structures or molecules are mainly governed by electrostatic interactions or hydrogen bonding due to the high electronegativity character of its surface [bib_ref] The chemistry of graphene oxide, Dreyer [/bib_ref]. This feature provides a very hydrophilic nature to GO, which was successfully exploited for the development of humidity sensors with unprecedented response speed [bib_ref] Ultrafast graphene oxide humidity sensors, Borini [/bib_ref]. Furthermore, GO was also explored as platform for the establishment of non-covalent bonding with DNA or proteins that allows the biorecognition events during biosensing [bib_ref] A graphene platform for sensing biomolecules, Lu [/bib_ref]. In case of graphene, although covalent strategies can be more efficient and stable they request dramatically changes on the structure by the disruption of the C=C bonds leading to the alteration of its characteristic electronic properties. For this reason, non-covalent functionalization of graphene is considered a simple and versatile method, offering the opportunity to attach different functionalities while simultaneously maintaining the integrity of the C-C sp 2 -hybridized carbon network. That fact offers a number of unique physicochemical properties that are desirable and advantageous for sensing applications. Pristine graphene possesses an extensive aromatic surface that allows the molecular interaction via π-π stacking and due to the atomic thickness the electric, mechanic and optical properties are highly sensitive to adsorbed molecules. Recently, the optoelectronic properties of graphene were explored as a biosensor for chemically specific label-free detection of proteins [bib_ref] Mid-infrared plasmonic biosensing with graphene, Rodrigo [/bib_ref]. The electromagnetic fields of graphene infrared (IR) plasmons display unprecedented spatial confinement which allows its using for enhanced light-matter interactions and integrated mid-IR photonics. The large mismatch between mid-IR wavelengths and biomolecules dimensions causing weak vibrational absorption signals is overcome by exploiting the presence of graphene plasmons near to biomolecules. Tunable plasmon resonance of nanostructured grapheme allows biomolecules high sensitivity by the extraction of its complex refractive index and the identification of vibrational fingerprints [bib_ref] Mid-infrared plasmonic biosensing with graphene, Rodrigo [/bib_ref]. For example, an interesting work showed that the functionalization of graphene with thionine by π-π stacking at the surface of glass carbon electrode allows the development of electrochemical sensor that can selectively recognize polycyclic aromatic hydrocarbons with high sensitivity and low detection limit [bib_ref] Graphene sheets, polyaniline and aunps based dna sensor for electrochemical determination of..., Wang [/bib_ref].
Reduced Graphene Oxide as explained before results from the partial recovery of aromatic structure through the gradual reduction of GO, showing an hybrid structure between sp 2 /sp 3 carbon regions on a global network [bib_ref] The chemistry of graphene oxide, Dreyer [/bib_ref]. rGO offers a large panoply of non-covalent bonding possibilities, being the degree of oxidation of those materials that balance the preferential chemical bonds that it can establish: π-π stacking, electrostatic interactions or hydrogen bonding. This material has the particular ability for the establishment of oxidation/reduction reactions with different molecules or structures. The electrical properties of rGO have been previously shown to be very sensitive to surface adsorbates, thus making rGO a very promising platform for highly sensitive electrochemical sensors [bib_ref] Recent trends in carbon nanomaterial-based electrochemical sensors for biomolecules: A review, Yang [/bib_ref] , molecular sensors [bib_ref] Reduced graphene oxide molecular sensors, Robinson [/bib_ref] and gas sensors [bib_ref] Highly selective gas sensor arrays based on thermally reduced graphene oxide, Lipatov [/bib_ref]. Despite all advantages of rGO for sensing applications, rGO-based gas sensors have poor selectivity because different gas molecules could be adsorbed on its surface and promote changes on the conductivity. Recently, the functionalization of rGO sheets through π-π stacking with tetra-α-iso-pentyloxyphthalocyanine showed an improved sensing performance to NH 3 gases at room temperature in comparison to that of pure rGO [bib_ref] Enhanced nh3-sensitivity of reduced graphene oxide modified by tetra-alpha-iso-pentyloxymetallophthalocyanine derivatives, Li [/bib_ref]. The enhanced sensing properties are attributed to the synergistic effect in rGO hybrids, with strong electron transfer interaction, superior electrical conductivity, and gas adsorption activity. Many other examples of the increase selectivity of rGO based materials through non-covalent interactions can be found in literature [bib_ref] Electrochemistry of graphene and related materials, Ambrosi [/bib_ref].
## Graphene nanoparticles hybrid materials
The closest integration of nanoparticles on graphene based materials surface are of a particular interest because it offer the possibility to obtain the individual properties of each element in a single material and at same time exhibit additional synergistic effects that can be very important on sensor development through the development of new sensing mechanism or increasing the sensitivity and selectivity of the system. The functionalization of graphene, GO and rGO with nanoparticles can occur by both covalent and noncovalent interactions using many different synthetic routes: growing the nanoparticles on their surface (in situ) or by attaching pre-synthesized nanoparticles to the graphene surface (ex situ) [bib_ref] Design, synthesis, and characterization of graphene-nanoparticle hybrid materials for bioapplications, Yin [/bib_ref]. However, GO and rGO are preferential substrates for the nucleation and growth of inorganic nanoparticles due to the high level of oxygen function groups and structural defects. Several hybrid graphene based materials have been described on literature using gold, silver, titanium dioxide, and iron oxide and nickel nanoparticles for sensor applications [bib_ref] Design, synthesis, and characterization of graphene-nanoparticle hybrid materials for bioapplications, Yin [/bib_ref]. The majority of graphene nanoparticle hybrid sensors developed are based on the electronic, electrochemical, and optical sensing mechanisms [bib_ref] Molecularly engineered graphene surfaces for sensing applications: A review, Liu [/bib_ref]. Nanoparticles can be useful for sensing system in many different ways: enhance surface area for detection, preserve the electrical properties of graphene by conjugating the probe, immobilization of biomolecules, catalyse electrochemical reactions, act as a reactant, double-quenching and dual enhancement of Raman signals via chemical and electromagnetic interactions. A recent review describes in detail the current progress that has been made in the development and application of graphene-nanoparticle hybrid sensors [bib_ref] Prospects for graphene-nanoparticle-based hybrid sensors, Yin [/bib_ref]. The greatest challenge that this technology faces at the moment consists on the fabrication of graphene-nanoparticle hybrid sensors able to produce sensing results in a reproducible way.
## Biosensing cancer biomolecules or cells
In a near future, biosensors are expected to detect with high sensitivity and selectivity a wide range of cancer biomarkers present in body fluids or overexpressed on cancer cells surface in order to diagnosis and monitoring the evolution of the disease during the treatment. In this way, biosensing systems employing the properties of graphene derivatives for cancer detection have found their way into a wide variety of strategies capable of improving the sensing efficiency of cancer biomarkers. In the following sections, we describe the application of graphene in optical imaging methods such as photoluminescence and Raman imaging, electrochemical sensors for enzymatic biosensing, DNA sensing, and immunosensing.
The changes that occur in the DNA sequence of the genomes of cancer cells are the reason of cancers arising [bib_ref] The cancer genome, Stratton [/bib_ref]. The profound metabolic remodeling of cancer cells, including mitochondrial rearrangement, is an indirect response to cell survival or proliferation, being controlled by specific cell signaling [bib_ref] Regulating Mitochondrial Respiration in Cancer, Serafim [/bib_ref]. The monitoring of molecular and physical events in live cells by visualization represent a key approach to understanding cell biology and have a profound influence on biomedical sciences progress. Biomedical sensors that can detect selective signals for biological molecules or physical variables in live cells with spatiotemporal resolution are a constant need. Fluorescent spectroscopic techniques offer sensing for intracellular signaling and analysis due to their non-invasiveness and high sensitivity [bib_ref] Nanomaterial-based fluorescent probes for live-cell imaging, Li [/bib_ref]. These optical features are of particular interest in the detection of cancer cells because they allow a sub-cellular resolution and therefore the capability of identifying tumor phenotypes at their very early stages [bib_ref] Two-photon and second harmonic microscopy in clinical and translational cancer research, Perry [/bib_ref]. Although the organic dyes and fluorescent proteins are powerful molecular probes, they present limitations in making reliable intracellular measurements because of their poor photobleaching resistance [bib_ref] Nanomaterial-based fluorescent probes for live-cell imaging, Li [/bib_ref]. In addition, their broad emission spectra may hamper practical applications since different fluorophores require multiple excitation wavelengths [bib_ref] Plasmonic nanoprobes for intracellular sensing and imaging, Yuan [/bib_ref].
The possible chemical interactions of this type of powerful molecular probes or steric hindrance with biomolecules may cause biotoxicity or perturbation to the systems being investigated which effect is not conceivable in medical applications [bib_ref] Sensing with fluorescent nanoparticles, Baù [/bib_ref].
## Photoluminescence
The development of nanoparticles with multifunctionalities has been a research focus in the last decade including graphene and its derivatives [bib_ref] A review of optical imaging and therapy using nanosized graphene and graphene..., Li [/bib_ref]. The unprecedented characteristics of these nanomaterials lead the researchers to suggest it as a new class of fluorescent probes for biomedical imaging. NG is photostable, non-toxic and easily conjugable [bib_ref] A review of optical imaging and therapy using nanosized graphene and graphene..., Li [/bib_ref] [bib_ref] Graphene-based nanomaterials for versatile imaging studies, Yoo [/bib_ref]. NG and NG derivatives can be used as fluorescence probes in photoluminescence imaging. Additionally, photoluminescence studies demonstrate that these carbon fluorescent probes may have two functionalities: specific detection of cancer cells and therapeutic agent (drug/gene vehicle or photodynamic therapy agent), that is present both cancer imaging and therapeutic functionalities [bib_ref] Target delivery and cell imaging using hyaluronic acid-functionalized graphene quantum dots, Al [/bib_ref] [bib_ref] Tracking the intracellular drug release from graphene oxide using surface-enhanced raman spectroscopy, Huang [/bib_ref] [bib_ref] Sers-fluorescence monitored drug release of a redox-responsive nanocarrier based on graphene oxide..., Chen [/bib_ref].
The high contrast bio-imaging produced by NG in the detection of cancer cells was demonstrated in the work of Peng et al. [bib_ref] Graphene quantum dots derived from carbon fibers, Peng [/bib_ref] The green NG was used to enhance the contrast visualization of the cells compounds. The nucleus of human breast cancer cell lines T47D were stained with DAPI and show blue color under imaging. After staining, cancer cells were treated with green NG during 4 h of incubation. The photoluminescence of GO was first used for cellular imaging by Dai et al. [bib_ref] Nano-graphene oxide for cellular imaging and drug delivery, Sun [/bib_ref] NGO was pegylated and conjugated covalently with a B-cell specific antibody Rituxan (anti-CD20) for selective binding to B-cell lymphoma cells. Since NGO showed luminescence in the visible and near infrared (NIR) regions the optical identification of cancer cells was possible. Additionally, NGO showed a dual functionality as drug carrier for Doxorubicin (DOX) and contrast agent for cancer cell imaging.
In vivo studies using NG prepared by a simple hydrothermal method with polythiophenes as the precursors indicated that NG can act as a multifunctional nanoplatform for the simultaneous imaging and highly efficient in photodynamic therapy (PDT) of cancer [bib_ref] A graphene quantum dot photodynamic therapy agent with high singlet oxygen generation, Ge [/bib_ref]. Tumor-targeting ligands such as folic acid (FA) [bib_ref] Multifunctional graphene quantum dots for simultaneous targeted cellular imaging and drug delivery, Wang [/bib_ref] and hyaluronic acid (HAu) [bib_ref] Target delivery and cell imaging using hyaluronic acid-functionalized graphene quantum dots, Al [/bib_ref] could be easily coupled onto NG for tumor targeting. Wang et al. [bib_ref] Multifunctional graphene quantum dots for simultaneous targeted cellular imaging and drug delivery, Wang [/bib_ref] developed a study where NG-FA was incubated with three different cell types that express FA receptor at different levels to demonstrate that FA preserves its binding activity to FA receptor even after its conjugation with NG. It was possible to observe that the fluorescence of NG in the human cervical carcinoma cell line HeLa is considerably stronger than in the adenocarcinomic human alveolar basal epithelial cell line and human embryonic kidney cell line HEK293A. In this study, the authors also proved that the green fluorescence arises from the internalization of NG-FA. Moreover, the results reveal the specific internalization of NG-FA by HeLa cells, consistent with the fact that HeLa cells overexpress FA receptor while A549 and HEK239A express FA receptor at a low level, indicating that NG-FA is internalized via endocytosis induce by FA receptor.
The hyaluronic acid was conjugated with NG by Abdullah-Al-Nahain et al. [bib_ref] Target delivery and cell imaging using hyaluronic acid-functionalized graphene quantum dots, Al [/bib_ref] The nanographene-hyaluronic acid (NG-HA) nanocomposite was tested on both mammalian cell lines MDCK (CD44 negative) and A549 of lungcarcinoma with CD44 overexpressed. The CD44 antigen is expressed in a large number of mammalian cells, it is a receptor for hyaluronic acid and participate in a wide variety of cellular functions, including tumor metastasis. Using findings from the confocal and cellular uptake investigation, it is clearly indicated that a larger amount of NG-HA was taken up by A549 cancer cells. On contrary, small amounts of non-targeted NG were allowed to enter into both types of cells, in which due to the presence of HA more NG-HA entered the cell cytoplasm via receptor-mediated endocytosis-based target delivery. The in vivo biodistribution and tumor specific delivery of NG-HA were also studied by imaging fluorescence. To investigate the efficient delivery The photoluminescence of GO was first used for cellular imaging by Dai et al. [bib_ref] Nano-graphene oxide for cellular imaging and drug delivery, Sun [/bib_ref] NGO was pegylated and conjugated covalently with a B-cell specific antibody Rituxan (anti-CD20) for selective binding to B-cell lymphoma cells. Since NGO showed luminescence in the visible and near infrared (NIR) regions the optical identification of cancer cells was possible. Additionally, NGO showed a dual functionality as drug carrier for Doxorubicin (DOX) and contrast agent for cancer cell imaging.
In vivo studies using NG prepared by a simple hydrothermal method with polythiophenes as the precursors indicated that NG can act as a multifunctional nanoplatform for the simultaneous imaging and highly efficient in photodynamic therapy (PDT) of cancer [bib_ref] A graphene quantum dot photodynamic therapy agent with high singlet oxygen generation, Ge [/bib_ref]. Tumor-targeting ligands such as folic acid (FA) [bib_ref] Multifunctional graphene quantum dots for simultaneous targeted cellular imaging and drug delivery, Wang [/bib_ref] and hyaluronic acid (HAu) [bib_ref] Target delivery and cell imaging using hyaluronic acid-functionalized graphene quantum dots, Al [/bib_ref] could be easily coupled onto NG for tumor targeting. Wang et al. [bib_ref] Multifunctional graphene quantum dots for simultaneous targeted cellular imaging and drug delivery, Wang [/bib_ref] developed a study where NG-FA was incubated with three different cell types that express FA receptor at different levels to demonstrate that FA preserves its binding activity to FA receptor even after its conjugation with NG. It was possible to observe that the fluorescence of NG in the human cervical carcinoma cell line HeLa is considerably stronger than in the adenocarcinomic human alveolar basal epithelial cell line and human embryonic kidney cell line HEK293A. In this study, the authors also proved that the green fluorescence arises from the internalization of NG-FA. Moreover, the results reveal the specific internalization of NG-FA by HeLa cells, consistent with the fact that HeLa cells overexpress FA receptor while A549 and HEK239A express FA receptor at a low level, indicating that NG-FA is internalized via endocytosis induce by FA receptor.
The hyaluronic acid was conjugated with NG by Abdullah-Al-Nahain et al. [bib_ref] Target delivery and cell imaging using hyaluronic acid-functionalized graphene quantum dots, Al [/bib_ref] The nanographene-hyaluronic acid (NG-HA) nanocomposite was tested on both mammalian cell lines MDCK (CD44 negative) and A549 of lungcarcinoma with CD44 overexpressed. The CD44 antigen is expressed in a large number of mammalian cells, it is a receptor for hyaluronic acid and participate in a wide variety of cellular functions, including tumor metastasis. Using findings from the confocal and cellular uptake investigation, it is clearly indicated that a larger amount of NG-HA was taken up by A549 cancer cells. On contrary, small amounts of non-targeted NG were allowed to enter into both types of cells, in which due to the presence of HA more NG-HA entered the cell cytoplasm via receptor-mediated endocytosis-based target delivery. The in vivo biodistribution and tumor specific delivery of NG-HA were also studied by imaging fluorescence. To investigate the efficient delivery of NG, the authors have used balb/c mice with 6 weeks of age. The tumors were grown, during 10 days (volume of 100mm3) on the back of the mice using A549 cells. to tumor tissue and its biodistribution, NG-HA was injected intravenously (10 mg/kg of body weight) through the tail vein. After 2 h, the mice with tumors were sacrificed and their organs dissected. The strong fluorescence from the tumors indicates the targeted accumulation of NG-HA to the tumor site. The biodistribution profiles of NG-HA showed that this nanocomposite was significantly accumulate in the tumor tissue due to the large number of leaky blood vessels surrounding the tumor. NG-HA was also accumulated in great quantities in liver and kidney tissue: blood circulation pass through liver and NG was uptake by the reticuloendothelial system and the accumulation of the kidney indicates a rapid excretion of this nanocomposite. Once again, the fluorescent NG-HA was also used to deliver DOX as treatment to cancer cells. The results showed that 60% was released within 12 h under the studied conditions. Moreover, it was found that almost all drugs were released within 48 h from the NG-HA matrix. of NG, the authors have used balb/c mice with 6 weeks of age. The tumors were grown, during 10 days (volume of 100mm3) on the back of the mice using A549 cells. to tumor tissue and its biodistribution, NG-HA was injected intravenously (10 mg/kg of body weight) through the tail vein. After 2 h, the mice with tumors were sacrificed and their organs dissected. The strong fluorescence from the tumors indicates the targeted accumulation of NG-HA to the tumor site. The biodistribution profiles of NG-HA showed that this nanocomposite was significantly accumulate in the tumor tissue due to the large number of leaky blood vessels surrounding the tumor. NG-HA was also accumulated in great quantities in liver and kidney tissue: blood circulation pass through liver and NG was uptake by the reticuloendothelial system and the accumulation of the kidney indicates a rapid excretion of this nanocomposite. Once again, the fluorescent NG-HA was also used to deliver DOX as treatment to cancer cells. The results showed that 60% was released within 12 h under the studied conditions. Moreover, it was found that almost all drugs were released within 48 h from the NG-HA matrix. In addition to tumor cell detection and recognition, NG can also be useful to monitor biochemical pathways, such as apoptosis, the programmed cell death that is a way for multicellular animal dispose of unwanted and damaged cells. The improper regulation of apoptosis has been associated to cancer, neurodegenerative and cardiovascular diseases [bib_ref] Apoptosis in neurodegenerative disorders, Mattson [/bib_ref] [bib_ref] Targeting programmed cell death in neurodegenerative diseases, Vila [/bib_ref]. Nonetheless, how the abnormal cell apoptosis begins and progress in vivo is not yet completely understood. The understanding of the various forms of apoptosis could be the key to unlocking the diagnosis and therapy of the related diseases. Numerous methods have been developed for imaging apoptotic cells in vitro [bib_ref] Nuclear apoptosis detection by flow cytometry: Influence of endogenous endonucleases, Lecoeur [/bib_ref] [bib_ref] Identification of programmed cell death in situ via specific labeling of nuclear..., Gavrieli [/bib_ref] , however there are few methods available for imaging apoptotic cells in live animals.
Recently, Roy et al. developed a novel method using NG modified with Annexin V antibody (AbA5) to form (AbA5)-modified NG (AbA5-NG) enabling to label apoptotic cells in live zebrafish (Danio rerio) [bib_ref] Photoluminescent graphene quantum dots for in vivo imaging of apoptotic cells, Roy [/bib_ref]. Zebrafish showed bright red photoluminescence in the presence of apoptotic cells. The fluorescent images of normal cells (MCF-10A cells) and cancer cells (HeLa and MCF-7 cells) after treatment with NG (2 mg¨mL´1) were separately recorded. It was possible to observe that the photoluminescent spots were distributed around the cell membranes and cytoplasm suggesting that NG penetrated into the cells via receptor-mediated and/or nonreceptor-mediated endocytosis. This study opens a novel opportunity to visualize the initiation and progression of apoptosis which might help in understanding the apoptotic mechanism better which is of major importance in diagnosis and treatment of cancer.
## Surface enhanced raman scattering
In the past few years the first reports of the feasibility of in vivo imaging of cancer with biocompatible surface-enhanced Raman scattering (SERS) probes have emerged although its application is traditionally in vitro. Fluorescence imaging is the most common bioimaging technique [bib_ref] Graphene-based nanomaterials for versatile imaging studies, Yoo [/bib_ref] [bib_ref] Graphene oxide based surface-enhanced raman scattering probes for cancer cell imaging, Liu [/bib_ref] [bib_ref] Raman microscopy for cellular investigations-from single cell imaging to drug carrier uptake..., Kann [/bib_ref] for the reasons mentioned in the previous sub-section but various intrinsic limitations including photobleaching due to unstable fluorescent dyes, autofluorescence from the biological samples, or limited multispectral detection owing to the spectral overlap between broadband fluorescence spectra hinders the further applications of fluorescence microscopy imaging [bib_ref] Autofluorescence of viable cultured mammalian cells, Aubin [/bib_ref] [bib_ref] Photobleaching kinetics of fluorescein in quantitative fluorescence microscopy, Song [/bib_ref]. In contrast, Raman scattering provides narrow spectral bandwidth and is resistant to photobleaching and autofluorescence and suitable for long-term monitoring of cellular processes [bib_ref] Noninvasive raman spectroscopy in living mice for evaluation of tumor targeting with..., Zavaleta [/bib_ref] [bib_ref] Real-time monitoring of glutathione-triggered thiopurine anticancer drug release in live cells investigated..., Ock [/bib_ref]. The low Raman scattering of an analyte, especially of biological nature, can be strongly enhanced by locating it near the surface of a noble metal nanostructure. This process known as surface-enhanced Raman scattering (SERS) makes that the amplification of the received signal overcomes the limitation of the intrinsically low Raman scattering efficiency of Raman spectroscopy [bib_ref] Raman microscopy for cellular investigations-from single cell imaging to drug carrier uptake..., Kann [/bib_ref]. SERS is capable of enhancing the Raman signals of analytes located near the surfaces of noble metal nanostructures by up to 10 orders of magnitude [bib_ref] Graphene oxide based surface-enhanced raman scattering probes for cancer cell imaging, Liu [/bib_ref] [bib_ref] Surface enhanced raman scattering enhancement factors: A comprehensive study, Ru [/bib_ref]. This allows the probing and imaging of cells with high sensitivity using SERS labels [bib_ref] Silver-gold core-shell nanoparticles containing methylene blue as sers labels for probing and..., Guo [/bib_ref] [bib_ref] Ultrasensitive near-infrared raman reporters for sers-based in vivo cancer detection, Samanta [/bib_ref] [bib_ref] Surface-enhanced raman spectroscopy: A new modality for cancer imaging, Andreou [/bib_ref].
Graphene oxide is an excellent candidate for biomedical imaging due to its inherent fluorescence and Raman scattering activity [bib_ref] Raman microscopy for cellular investigations-from single cell imaging to drug carrier uptake..., Kann [/bib_ref]. GO and metal nanoparticle nanocomposites have also been used for diverse SERS applications in cellular imaging. Gold and silver nanoparticles are the most common used in SERS technique and for such reason are strong candidates to perform GO/metal nanocomposites. The presence of metallic nanoparticles enhances Raman characteristic bands, moreover, GO in biological media acts as a fluorescence quencher. These features indicate that GO/metal nanocomposites with specific binders may act as signaling when inside the cancer cells.
Hereafter the potentialities of graphene nanocomposites in the detection and, in some cases, therapeutic agent using SERS technique will be discussed.
GO decorated with Au nanoparticles were internalized into individual cells to provide localized sensing at the subcellular level [bib_ref] Cell imaging by graphene oxide based on surface enhanced raman scattering, Liu [/bib_ref]. The sensitivity was remarkably improved and the acquisition time was effectively shortened as a result of the SERS effect. The cellular uptake mechanism of GO was also investigated. Hella 229 cells were incubated in a medium containing either GO or GO/Au nanocomposites at 4˝C and 37˝C for 2 h respectively. It could be seen that different parts within the cells exhibited different Raman intensities. In the Raman images of the cells [fig_ref] Figure 4: The cellular uptake mechanism investigations of GO and Au/GO hybrids [/fig_ref] , the dark regions correspond to the cell nucleus. The authors concluded that neither GO nor GO/Au went through the nucleus membrane and thus they mainly reside in cytoplasm.
Sensors 2016, 16, x 10 of 28 cells [fig_ref] Figure 4: The cellular uptake mechanism investigations of GO and Au/GO hybrids [/fig_ref] , the dark regions correspond to the cell nucleus. The authors concluded that neither GO nor GO/Au went through the nucleus membrane and thus they mainly reside in cytoplasm. The study of the mechanism of the cellular uptake was also performed by Huang et al. [bib_ref] Mechanism of cellular uptake of graphene oxide studied by surface-enhanced raman spectroscopy, Huang [/bib_ref] , using GO loaded with Au nanoparticles as SERS substrate. In this work, once again it was proven the importance of the graphene nanocomposite in the detection of cancer cells besides its contribution on the knowledge about the cellular mechanism uptake. To understand the cell internalization process of the GO/Au, SERS spectra of GO/Au incubated with Ca Ski cells for 1, 2, 4, 6, 8, and 12 h were measured. The strongest SERS signals were recorded after 6 h of incubation. After that, the SERS signal weakened, and was just above the noise level at 12 h of incubation. These results may indicate that, under the present experiments conditions, the amount of GO/Au taken up by Ca Ski cells increased significantly at an incubation time of 4 h, and reached a maximum at 6 h, being the Au nanoparticles released from the GO sheet in the cell environment, after this incubation time.
Manikandan et al. have proposed several SERS substrates based on the nucleation of gold nanohexagons (Au) on graphene nanosheets [bib_ref] Facile synthesis of gold nanohexagons on graphene templates in raman spectroscopy for..., Manikandan [/bib_ref]. These were applied to enhance Raman scattering and differentiate human breast normal cancer cells and cancer stem cells. These SERS substrates led to 5.4 fold increase the detection of breast cancer cells and 4.8 fold of sensitivity for detecting breast cancer stem cells. This study demonstrates the potential of graphene to differentiate between normal cells, cancer cells and cancer stem cells by SERS which is considered as the primary tumor focus.
A gold capture substrate modified with linker molecules and antibodies, covered by a graphene monolayer, was developed by Krasnoslobodtsev et al. [bib_ref] Nano-immunoassay with improved performance for detection of cancer biomarkers, Krasnoslobodtsev [/bib_ref]. In this work, pancreatic cancer cells presented the mucin protein (MUC4) overexpressed, which was used as a biomarker in serum-based assay for early diagnosis of such type of tumor. As MUC4 was not detected in serum by the conventional bioassays used in clinical settings as enzyme linked immunosorbent assay (ELISA) and radioimmunoassay (RIA), the authors prepared a SERS substrate for MUC4 sandwich immunoassay The study of the mechanism of the cellular uptake was also performed by Huang et al. [bib_ref] Mechanism of cellular uptake of graphene oxide studied by surface-enhanced raman spectroscopy, Huang [/bib_ref] , using GO loaded with Au nanoparticles as SERS substrate. In this work, once again it was proven the importance of the graphene nanocomposite in the detection of cancer cells besides its contribution on the knowledge about the cellular mechanism uptake. To understand the cell internalization process of the GO/Au, SERS spectra of GO/Au incubated with Ca Ski cells for 1, 2, 4, 6, 8, and 12 h were measured. The strongest SERS signals were recorded after 6 h of incubation. After that, the SERS signal weakened, and was just above the noise level at 12 h of incubation. These results may indicate that, under the present experiments conditions, the amount of GO/Au taken up by Ca Ski cells increased significantly at an incubation time of 4 h, and reached a maximum at 6 h, being the Au nanoparticles released from the GO sheet in the cell environment, after this incubation time.
Manikandan et al. have proposed several SERS substrates based on the nucleation of gold nanohexagons (Au) on graphene nanosheets [bib_ref] Facile synthesis of gold nanohexagons on graphene templates in raman spectroscopy for..., Manikandan [/bib_ref]. These were applied to enhance Raman scattering and differentiate human breast normal cancer cells and cancer stem cells. These SERS substrates led to 5.4 fold increase the detection of breast cancer cells and 4.8 fold of sensitivity for detecting breast cancer stem cells. This study demonstrates the potential of graphene to differentiate between normal cells, cancer cells and cancer stem cells by SERS which is considered as the primary tumor focus.
A gold capture substrate modified with linker molecules and antibodies, covered by a graphene monolayer, was developed by Krasnoslobodtsev et al. [bib_ref] Nano-immunoassay with improved performance for detection of cancer biomarkers, Krasnoslobodtsev [/bib_ref]. In this work, pancreatic cancer cells presented the mucin protein (MUC4) overexpressed, which was used as a biomarker in serum-based assay for early diagnosis of such type of tumor. As MUC4 was not detected in serum by the conventional bioassays used in clinical settings as enzyme linked immunosorbent assay (ELISA) and radioimmunoassay (RIA), the authors prepared a SERS substrate for MUC4 sandwich immunoassay with a graphene protective monolayer. This SERS-based assay showed the ability to correctly detect and quantify low levels of MUC4, leading to an accurate identification of the cancer prostate patients.Silver nanoparticles have been considered as a superior SERS-active substrate with surface plasmon frequencies from the UV to the near-infrared region changing the ratio of its shell thickness with a core size [bib_ref] One-step synthesis of silver nanoshells with bumps for highly sensitive near-ir sers..., Kang [/bib_ref]. A SERS-active nanostructure was prepared by encapsulating Ag nanoshell (AgNS) with GO denoted by GONS. In this work, MCF-7 (human breast adenocarcinoma cell line) cells were treated with GONS. Before the treatment with GONS nanoprobes, the cells had no detectable signals; however, strong SERS signals clearly appeared in the cells that subjected to the treatment. The internalization of this type of nanoprobes shows a higher accumulation of GONS on the cell membrane, although a few were internalized into the cytoplasm. Thirty four different cells were analyzed by Raman spectroscopy and SERS signals of GONS nanoprobes were observed from 82% of the cells which indicates that this graphene-based system is an ultrasensitive nanoprobe for imaging of cancer cells.
Based on the natural high affinity of folate for the folate receptor protein (FR), which is commonly expressed on the surface of many human cancers, folate conjugates bind to the FR and causes the cellular uptake via endocytosis. The conjugation of the specific binding of folic acid (FA) and graphene-metal nanoparticles improve the targeted detection of cancer cells. Liu et al. [bib_ref] Graphene oxide based surface-enhanced raman scattering probes for cancer cell imaging, Liu [/bib_ref] prepared a SERS label based on GO/Ag with FA conjugated covalently. FR-positive HeLa (human cervical cancer) cells and FR-negative A549 (adenocarcinomic human alveolar basal epithelial) were incubated with GO/Ag-FA. Though both types of cells are carcinomic, the binding specificity of FA results in intense Raman signals on imaging of FR-positive cancer cells. A similar study was made by Hu and co-workers [bib_ref] Fabrication of reduced graphene oxide and sliver nanoparticle hybrids for raman detection..., Hu [/bib_ref] by using GO/Ag-FA nanocomposite to distinguish FRs over-expressed cancer cells from the cells on which the FRs are not over-expressed.
In order to apply SERS in real-world, the nucleosides excreted from normal human urine (n = 40) and from breast cancer patients (n = 40) were measured using a graphene-silver-copper nanoparticles (G/SCNP) as SERS probe [bib_ref] Growth graphene on silver-copper nanoparticles by chemical vapor deposition for high-performance surface-enhanced..., Zhang [/bib_ref]. On the basis of the obtained G/SCNPs, the SERS spectra of the nucleosides excreted from normal human and breast cancer patients reveal significant difference in some spectral ranges.
The chemotherapy is the most common cancer treatment but its huge side-effects make it very painful for the patients. It is urgent to promote effective and targeted deliver of drugs used in chemotherapy since they show good results. SERS can contribute to monitor such deliver using multifunctional nanoprobes. GO-based nanoplatform simultaneously loaded with chemical drug and Ag nanoparticles was prepared and employed to study the drug release from GO in living cancer model cells by SERS [bib_ref] Tracking the intracellular drug release from graphene oxide using surface-enhanced raman spectroscopy, Huang [/bib_ref]. In this work, DOX was loaded onto GO by electrostatic interaction and Ag nanoparticles were covalently linked onto GO. Real-time measurement of SERS signals of DOX using the GO loaded with Ag NPs as a SERS-active substrate allowed monitoring the process of the drug release inside the living cell. The SERS results revealed that DOX is initially released from the GO surface inside the lysosomes, then escapes into the cytoplasm, and finally enters the nucleus, while GO, the nanocarrier, remains within the cytoplasm, without entering the nucleus. The DOX release into cancer cells was also monitored by SERS imaging using a redox-responsive GO/Ag nanocarrier [bib_ref] Sers-fluorescence monitored drug release of a redox-responsive nanocarrier based on graphene oxide..., Chen [/bib_ref]. The drug was loaded to the surfaces of GO by disulfide linkages which can be cleaved by glutathione (GSH). Covalent drug loading and GSH-responsive release strategy can reduce the influence of the surface diffusion barriers introduced by multifunctionalization. Since tumor cells generally exhibit a higher concentration of GSH than normal ones, this drug carrier should have potential in the field of tumor therapy. GSH triggered drug release process was studied using HeLa cells as a model of tumor cells with a high GSH level. In the presented nanocarriers, the intrinsic Raman bands of NGO were enhanced by attaching Ag nanoparticles on the NGO surfaces, which were used to do SERS mapping images to express the distribution of nanocarriers inside HeLa cells. In addition, the real-time release dynamics were given by detection of SERS signals of DOX in situ inside HeLa cells. With such results it was clearly demonstrated that SERS offers distinct advantages over the widely used fluorescence technique in that it can provide direct information on the carrier and the drug inside the living cells simultaneously, and without dye labeling.
## Bioquantification of cancer biomarkers and cells
A biosensor is defined as an integrated device that is able of provides quantitative or semi-quantitative information about biomolecules or biostructures by using biological recognition element. The main difference relatively of a bioanalytical system is that requires additional processing steps such as addition of reagents in order to be able to quantify the desired analyte. The structure of a sensor is usually constituted by two key elements: the receptor and the transducer. The receptor should guaranty the specific interaction with the proper analytic or many different analytes and the transducer is responsible to convert the perturbation caused on the receptor (input) through the interaction with the analyte into a measurable signal (output).
Hereafter we will discuss particularly electrochemical methods such as voltammetry and amperometry which are generally adopted transducing techniques for the development of graphene based sensors for biosensing due to their simplicity, high sensitivity and low-cost. However sensors based on other detection methods like FRET, photoluminescence, fluorescence, optical and SERS start to arise with very promising results on the biodetection of cancer markers.
## Electrochemical sensors
## Immunosensors
Immunoassay has becoming one of the most important tools for clinical diagnosis of cancer. Detection of cancer biomarkers associated with certain tumors in early stage of the disease becomes on important contribute for the successful treatment. Graphene sensors promise to be an innovative technology in next generation electronics on cancer diagnostic applications, due to exceptional electronic properties and extreme surface to volume ratio able to offer greatly enhanced sensitivity for the detection of cancer biomarkers. New graphene-based technology allows the development of portable and rapid diagnostic sensors whit potential to overcome the features of conventional enzyme-linked immunobsorbant assays (ELISAs). Hereafter we describe the potential use of graphene and its derivatives on the development of new electrochemical sensors for detection of specific cancer biomarkers.
## Carcinoembryonic antigen
Carcinoembryonic antigen (CEA) is one of the most extensively used tumor markers on early detection of lung, ovarian carcinoma, breast cancers and cystadenocarcinoma. A novel electrochemical immunosensor based on carbon ionic liquid electrode (CILE) modified with AuNPs, rGO and poly(l-Arginine) nanocomposite material as sensitive platform for detection of CEA [bib_ref] Electrochemical immunoassay based on gold nanoparticles and reduced graphene oxide functionalized carbon..., Yu [/bib_ref]. The synergistic effects of nanocomposite resultant from the individual properties of each material allow an enhanced capacity of adsorption of CEA antibody and a consequently improvement of the electrochemical responses. At optimized conditions, differential pulse voltammetric responses were proportional to CEA concentration in the range from 0.5 to 200 ng¨mL´1 with the detection limit as 0.03 ng¨mL´1. Another work reported the development of a novel electrochemical immunosensor based on the deposition of ultrathin Au-Pt nanowire-decorated thionine/reduced graphene oxide (AuPtNWs/THI/rGO) on the surface of glass carbon electrode (GCE) [bib_ref] Large-scale synthesis of ultrathin au-pt nanowires assembled on thionine/graphene with high conductivity..., Lu [/bib_ref]. The differential pulse voltammetric responses were proportional to CEA concentration in the range from 50 fg¨mL´1 to 100 ng¨mL´1 with an detection of 6 fg¨mL´1. A very simple approach using GO-Thi-Au nanocomposites as sensing platform for the surface modification of GCE was been successfully employed for the fabrication of ultrasensitive electrochemical immunosensor for CEA [bib_ref] One-step synthesis of graphene oxide-thionine-au nanocomposites and its application for electrochemical immunosensing, Han [/bib_ref]. The immunosensor showed a linear calibration plot in a wide concentration range from 0.1 fg¨mL´1 to 1ˆ10 9 fg¨mL´1 and ultralow detection limit of 0.05 fg¨mL´1.
Electrochemical immunosensors based on Au electrodes modified with graphene nanocomposites as a sensing platform showed to be also a viable approach for the detention of CEA. Samanman et al. reported the Au electrode surface modification by deposition of a thin film of silver and then with a AuNPs-rGO-chitosan nanocomposite prepared by cryogel functionalized with anti-CEA [bib_ref] Highly-sensitive label-free electrochemical carcinoembryonic antigen immunosensor based on a novel au nanoparticles-graphene-chitosan..., Samanman [/bib_ref]. At optimal conditions, the decrease of the cyclic voltammetry silver peak current was proportional to the CEA concentration over a range of from 1.0 fg to 1.0 ng¨mL´1 with a detection limit of 0.1 fg¨mL´1. In another work Au electrode surface was initially modified with rGO-SWCNTs-Chitosan nanocomposite (Chi-rGO-CNTs) followed by electrodeposition with Au and Pt NPs and then functionalized with anti-CEA. The proposed immunosensor showed CEA determination of range 0.1 pg¨mL´1 to 40 ng¨mL´1 with a limit of detection down to 30 fg¨mL´1 [bib_ref] An ultrasensitive luminol cathodic electrochemiluminescence immunosensor based on glucose oxidase and nanocomposites:..., Jiang [/bib_ref]. Graphene biosensors based on sandwich-type immunosensors methods were also been extensively explored on the detection of CEA. High-affinity antibodies and appropriate labels are usually employed for the amplification of detectable signal [bib_ref] Sandwich-type immunosensors and immunoassays exploiting nanostructure labels: A review, Pei [/bib_ref]. Recent research has looked to develop innovative and powerful novel graphene labels or graphene sensing platforms, controlling and tailoring their properties in a very predictable manner to meet the requirements for increasing the detection level and specificity for cancer biomarker. One of the pioneering work, reported a sandwich luminol electrochemiluminescence (ECL) immunosensor using ZnO nanoparticles (ZnONPs) and glucose oxidase (GOD) decorated rGO as labels and in situ generated hydrogen peroxide as coreactant (ZnONPs@rGO-GOD-Ab2) [bib_ref] Highly sensitive luminol electrochemiluminescence immunosensor based on ZnO nanoparticles and glucose oxidase..., Cheng [/bib_ref]. The sensing platform at GCE electrode surface was obtained by the assembly with a hybrid architecture of AuNPs/rGO able to provide high immobilization sites for antibody (Ab1). After the biorecognition event of CEA the ZnONPs@rGO-GOD-Ab2 labels were able to be immobilized on the electrode surface (GCE/rGO-AuNPs/Ab1) via sandwich immunoreactions. Enhanced sensitivity was obtained by in situ generating hydrogen peroxide with glucose oxidase and the catalysis of ZnONPs to the ECL reaction of luminol-H 2 O 2 system. The rGO-based immunosensor exhibited a detection range of CEA from 10 pg¨mL´1 to 80 ng¨mL´1 and a detection limit of 0.0033 ng¨mL´1. Recently, another work describes the use of rGO on both sensor sensing platform and as a signal amplifying factor for construction of the "sandwich-type" immunosensor with ultrahigh efficiency for CEA detection [bib_ref] Ag/au nanoparticles coated graphene electrochemical sensor for ultrasensitive analysis of carcinoembryonic antigen..., Huang [/bib_ref]. Graphene sensing platform was prepared by π-π stacking with 1,5-diaminonaphthalene (DN) an then coated with AuNPs conjugated with antigen CEA (Ab1). Graphene tracer label was also based on graphene/DN composite coated with Ag/Au nanoparticles and conjugated with antigen (Ab2). The biorecognition event occurs after the addition of CEA, by immune reactions of antigens and antibodies, which results in the great current signal owing to the existence of electroactive element Ag/AuNPs. The cyclic voltammetric results showed a linear increased of current with the increasing antigen CEA concentrations in the range of 10 to 1.2ˆ10 5 pg¨mL´1 and a limit of detection 8 pg¨mL´1.
Graphene made by CVD was also explored as electrochemical sensing platform, in the form of high surface area conducting electrodes surfaces for the high sensitive detection of CEA. The graphene sensing surface was modified with magnetic beads (MBs) functionalized with CEA detection antibody (Gr/MBs-Ab1), by applying an external magnetic field. In order to enhance the sensitivity of the sensor, AuNPs were modified with horse radish peroxidase (HRP) and detection antibody (Ab2), to form the conjugate Ab2-AuNPs-HRP [bib_ref] Multi-nanomaterial electrochemical biosensor based on label-free graphene for detecting cancer biomarkers, Jin [/bib_ref]. The electrochemical immunosensor analysis was based on the biorecognition event with CEA, using GR/MBs-Ab1/CEA/Ab2-AuNPs-HRP system as a tracer and H 2 O 2 as an enzyme substrate, in a sandwich-type immunoassay format. Experimental results showed linear relationship between peak current and CEA concentration for the range of 5-60 ng¨mL´1. The limit of detection was 5 ng¨mL´1. Several works in the last year have been reported proposing new architectures for the development of sandwich-type graphene-based electrochemical immunosensor for CEA detection, where graphene can acts as sensing platform at electrode surface, label agent for the increase of sensitivity or by performing both activities. Numerous studies in the last year have been reported proposing new architectures for the development of sandwich-type graphene-based electrochemical immunosensor for CEA detection, where graphene can acts as sensing platform at electrode surface, conjugate agent for the increase of sensitivity or perform both activities. The performance of each sandwich-type graphene-based biosensor was compared in [fig_ref] Table 1: Sandwich-type graphene-based biosensor performance for the detection of carcinoembryonic antigen [/fig_ref].
## Gce/aunps-rgo-ab1
ZnONPs-rGO-GOD 0.01-80 0.0033 [bib_ref] Highly sensitive luminol electrochemiluminescence immunosensor based on ZnO nanoparticles and glucose oxidase..., Cheng [/bib_ref] Graphene/Magnetic Beads-Ab1 AuNPs-horse radish peroxidase (HRP)-Ab2 5-60 5 [bib_ref] Multi-nanomaterial electrochemical biosensor based on label-free graphene for detecting cancer biomarkers, Jin [/bib_ref] rGO/AuNPs-Ab1 Ag/AuNPs-rGO-Ab2 0.0001-200 0.008 [bib_ref] Ag/au nanoparticles coated graphene electrochemical sensor for ultrasensitive analysis of carcinoembryonic antigen..., Huang [/bib_ref] GCE modified with AuNPs-SWCNTs-rGO-Ab1
Pd/Pt-rGO-glucose oxidase (GOD)-Ab2 0.0001-160 3.0ˆ10´5 [bib_ref] Amplified cathodic electrochemiluminescence of luminol based on pd and pt nanoparticles and..., Cao [/bib_ref] Au electrode/rGO-Nanoporous Au-Ab1 AuNPs/PDDA-GS/MnO 2 -Ab2 0.0003-8 0.08 [bib_ref] Synthesis and characterization of graphene nanosheets attached to spiky mno2 nanospheres and..., Zhang [/bib_ref] Au electrode/AuNps-Ab1 PTC-Arg/Au@rGO complexes 0.001-10 0.
## [100] gce/aunps-rgo-hemin-ab1
AgNPs-rGO-GOD-Ab2 0.0001-160 3.0ˆ10´5 [bib_ref] Electrochemiluminescence of luminol enhanced by the synergetic catalysis of hemin and silver..., Jiang [/bib_ref] rGO-AuNPs modified GCE microfluidic paper analytical devices (µPADs) P-acid/Pt-Ag Alloy NPs-Ab2 0.001-100 0.0003 [bib_ref] An origami electrochemiluminescence immunosensor based on gold/graphene for specific, sensitive point-of-care testing..., Yan [/bib_ref] Prostate Specific Antigen
Prostate specific antigen (PSA) is a characteristic tumor marker of prostate cancer used in prostate cancer diagnosis and screening. The first attempt to use graphene for sensitive detection of PSA was trough the fabrication of a label-free electrochemical immunosensor. The sensor technology was based on the surface functionalization of GCE electrode with a thin film of the nanocomposite, rGO, 1-pyrenebutanoic acid, succinimidyl ester (PBSE) and CoNPs, as a sensing platform [bib_ref] Label-free electrochemical detection of cancer marker based on graphene-cobalt hexacyanoferrate nanocomposite, Li [/bib_ref]. The PBSE was conjugated with Anti-PSA antibody in order to increase the selectivity for PSA. The measurements suggested that electroactivity of CoNPs was greatly enhanced in the presence of rGO due to its great electron-transfer ability. The specific antibody-antigen immunocomplex formed on the electrode resulted in the decrease of amperometric signal, linearly dependent with PSA concentration in the range of 0.02-2 ng¨mL´1 with a low detection limit of 0.01 ng¨mL´1. Recently a new approach based on highly conductive crumpled like structure rGO/AuNPs nanocomposite as a sensing platform for detection of PSA was reported [bib_ref] 3D label-free prostate specific antigen (PSA) immunosensor based on graphene-gold composites, Jang [/bib_ref]. The 3D immunosensor showed a good linear relationship between the current change and different concentrations of PSA from 0-10 ng¨mL´1 and the detection limit 0.59 ng¨mL´1.
Recently a novel label electrochemical graphene-based immunosensor for PSA was reported. The sensing surface of the GCE was constructed based on the deposition of new amino-functionalized graphene sheet ferrocenecarboxaldehyde composite material (NH2-rGO@FCA) [bib_ref] Label electrochemical immunosensor for prostate-specific antigen based on graphene and silver hybridized..., Li [/bib_ref]. The sensor label was based on silver hybridized mesoporous silica NPs (Ag@NH 2 -MCM48), able to improve the electron transfer ability of the immunosensor system. The workability of immunosensors system Ag@NH 2 -MCM48/Ab2 and NH 2 -GS@FCA was based on the linear catalytic current increase with the PSA concentration. The immunosensor showed a good relationship between the current responses toward PSA concentration on the range from 0.01 to 10.0 ng¨mL´1 similar to the non-labeled ones, however the detection limit was as much lower 0.002 ng¨mL´1.
Carbohydrate Antigen 19-9 and 15-3
Carbohydrate Antigen 19-9 (CA 19-9) is not sensitive or specific to use as a screening test for cancer but can be used as a tumor marker if is produced in abnormal amounts. When CA 19-9 level is elevated to high concentrations it is often correlated to with gastrointestinal malignancies such as cholangiocarcinoma, pancreatic cancer, or colon cancer. Yang et al. proposed a novel sandwich-type graphene-based electrochemical immunosensor for the detection of CA19-9 [bib_ref] Ultrasensitive electrochemical immunosensor for carbohydrate antigen 19-9 using au/porous graphene nanocomposites as..., Yang [/bib_ref]. The sensing surface of GCE was built through the assembly Au-rGO nanocomposite and posterior immobilization of anti-CA19-9, forming the structure Ab1/Au-rGO/GCE. The signal enhancers were designed though the GO surface modification with core shell Au@Pd NPs, thionine (Thi), anti-CA19-9 (Ab2) and Horseradish peroxidase (HRP) forming signal probes Au@Pd-Gra/Thi-Ab2/HRP. During the biorecognition event, the target protein CA19-9 was sandwiched between the primary antibody Ab1/Au-rGO/GCE and the prepared bioconjugates Au@Pd-Gra/Thi-Ab2/HRP, resulting in enhancement of the detectable signal. The results showed that peak currents of the electrochemical immunoassay increased with the increase of CA19-9 concentrations, and exhibited a linear relationship range from 0.015 to 150 U¨mL´1, with low detection limit of 0.006 U¨mL´1.
Carbohydrate Antigen 15-3 (CA 15-3) is a tumor marker used to monitor certain cancers, particularly breast cancer. The first technological approach for the sensitive detection of CA 15-3 using graphene based materials consisted on the development of label-free electrochemical immunosensor [bib_ref] Highly selective gas sensor arrays based on thermally reduced graphene oxide, Lipatov [/bib_ref]. The immunosensor, was designed by the surface modification of GCE with a highly conductive N-doped graphene sheets, that allows a significantly increase of electron transfer and high sensitivity toward CA 15-3. The immunosensor exhibited a low detection limit at 0.012 U/mL and a broad linear response in the range of 0.1-20 U/mL. Recently, it was reported a novel electrochemical graphene based immunosensor for sensitive detection of CA15-3 based on dual signal amplification strategy [bib_ref] Ionic liquid functionalized graphene based immunosensor for sensitive detection of carbohydrate antigen..., Zhao [/bib_ref]. For trace tag signal amplifier was synthesized a nanocomposite material based on nanoporous TiO 2 functionalized with Cd 2+ for increase the electron transfer rate, followed by the immobilization of the antibody CA15-3 (Ab2), resulting in the following sequence (Ab(2)-f-TiO 2 -Cd 2+ ). The GCE sensing platform was developed by immobilization of rGO functionalized with ionic liquid and primary CA15-3 antibody (Ab(1)) resulting in the sequence GS-Ab(1). Through biorecognition reaction, the target protein CA15-3 was sandwiched between the primary antibody GS-Ab1 and the prepared (Ab2-f-TiO 2 -Cd 2+ ) bioconjugates, resulting in an enhanced detectable signal. The resultant immumosensor displayed a wide range of linear response (0.02-60 U/mL), ultra-low detection limit (0.008 U/mL), good reproducibility, selectivity and stability towards CA15-3.
## Protein p53
Protein p53 is a well-known tumor suppressor and a transcription factor fundamental in the control of cell growth and modulation of DNA repair processes [bib_ref] Regulating the p53 pathway: In vitro hypotheses, in vivo veritas, Toledo [/bib_ref]. Loss of p53 function caused by the conformational changes in p53 protein structure, results in an induction of tumors and gene mutation [bib_ref] Post-translational modification of p53 in tumorigenesis, Bode [/bib_ref]. Some clinical evidences showed the implication of p53 phosphorylation in human cancers, particularly expression of p53 protein phosphorylated, serine 15 (Ser15) serine 20 (Ser20) and serine 392 (Ser392). In that sense the quantitative analysis of phosphorylated p53 is crucial for early cancer diagnosis. Most of the electrochemical graphene-based immunosensors engineered for sensitive detection of phosphorylated p53, were based on sandwich type strategy. The fist graphene-based immunosensor reported was dedicated to the specific detection of phosphorylated p53 on serine 392. For immunosensor fabrication it was used screen-printed carbon electrode (SPCE) modified with AuNPs to self-assemble a layer of N-hydroxysuccinimideactivated hexa (ethylene glycol) undecane thiol (NHS) for primary attachment of phospho-p53392 capture antibody (Ab1/NHS/AuNPs-SPCE) [bib_ref] Functionalized graphene oxide as a nanocarrier in a multienzyme labeling amplification strategy..., Du [/bib_ref]. The HRP-p53392Ab2-GO conjugate was synthetized by the functionalization of carboxylated GO with HRP and the immobilization of capture antibody p53392 (Ab2) by amidization reaction. After sandwich immunoreaction, the HRP-p53392Ab2-GO captured onto the electrode surface produced an amplified electrocatalytic response by the reduction of enzymatically oxidized thionine in the presence of H 2 O 2 [fig_ref] Figure 5: Schematic Illustration of the Multi-enzyme Labeling Amplification Strategy Using HRP-p53392Ab2-GO Conjugate [/fig_ref]. The increase of response current was proportional to the phospho-p53392 concentration in the range of 0.02-2 nM with the detection limit of 0.01 nM.
Sensors 2016, 16, x 16 of 28 enzymatically oxidized thionine in the presence of H2O2 [fig_ref] Figure 5: Schematic Illustration of the Multi-enzyme Labeling Amplification Strategy Using HRP-p53392Ab2-GO Conjugate [/fig_ref]. The increase of response current was proportional to the phospho-p53392 concentration in the range of 0.02-2 nM with the detection limit of 0.01 nM. Another work reported the development of graphene-based immnunnosensor for specific detection of phosphorylated p53 on serine 15. In that case graphene was integrated on the sensing platform instead of the sandwich conjugate platform. For the fabrication of the immunosensor the SPCE was functionalized with graphene and chitosan for primary antibody attachment phospho-p5315 (Ab1/graphene-CHI/SPCE) [bib_ref] Graphene-based immunosensor for electrochemical quantification of phosphorylated p53 (s15), Xie [/bib_ref]. The sandwich immunocomplex was formed among phospho-p5315 capture antibody, phosphop5315 antigen, biotinylated phospho-p5315 detection antibody and HRP-labeled streptavidin, resulting in the following system: HRPstreptavidin-biotinAb2/phospho-p5315/Ab1/graphene-CHI/SPCE. The linear response was observed for concentration range of phospho-p5315 from 0.2 to 10 ng·mL −1 , with the detection limit of 0.1 ng·mL −1 .
## Vascular endothelial growth factor (vegf)
Vascular endothelial growth factor (VEGF) is an important growth factor that regulates the progression of angiogenesis. VEGF overexpression appears in many human cancers, such as brain tumors, lung cancer, breast cancer, gastrointestinal cancer and urinary tract tumors [bib_ref] The biology of vegf and its receptors, Ferrara [/bib_ref]. Lin et al. developed a simple and reusable strategy for detecting the VEGF concentration in human serum using a graphene based biosensor [bib_ref] A reusable magnetic graphene oxide-modified biosensor for vascular endothelial growth factor detection..., Lin [/bib_ref]. The sensing platform deposited at Au electrode surface consisted of a nanocomposite of GO-F3O4NPs functionalized with Avastin, as the specific biorecognition element for VEGF. The amperometric signal was measured by differential pulse voltammetry to quantify the VEGF concentration, being the linear response of the biosensensor observed for the range of VEGF concentrations from 0.03125 to 2 ng·mL −1 . The biosensors showed a good stability (RSD = 2.36%, n = 50) and a limit of detection of 0.03125 ng·mL −1 .
## Alpha-fetoprotein (afp)
Alpha-fetoprotein (AFP) is a glycoprotein of fetal component produced during the embryonic period. However, high AFP serum levels can be found mainly in patients with hepatocellular carcinoma or other types of cancer such as, lung cancer, biliary cancer, gastric cancer, pancreatic cancer, teratocarcinoma of the testis. Graphene-based materials have been explored as a sensing platform for the development of noble electrochemical immunoassay in order to amplify the sensitivity on detection of AFP. The pioneer work reported a graphene-based ultrasensitive multiplexed electrochemiluminescent (ECL) immunoassay method for the detection AFP tumor marker [bib_ref] Sandwich-type electrochemiluminescence immunosensor based on pdda-g@lu-au composite for alpha-fetoprotein detection, Gan [/bib_ref]. The graphene probe was synthesized by the assembly with poly Another work reported the development of graphene-based immnunnosensor for specific detection of phosphorylated p53 on serine 15. In that case graphene was integrated on the sensing platform instead of the sandwich conjugate platform. For the fabrication of the immunosensor the SPCE was functionalized with graphene and chitosan for primary antibody attachment phospho-p5315 (Ab1/graphene-CHI/SPCE) [bib_ref] Graphene-based immunosensor for electrochemical quantification of phosphorylated p53 (s15), Xie [/bib_ref]. The sandwich immunocomplex was formed among phospho-p5315 capture antibody, phosphop5315 antigen, biotinylated phospho-p5315 detection antibody and HRP-labeled streptavidin, resulting in the following system: HRP-streptavidin-biotinAb2/phospho-p5315/Ab1/graphene-CHI/SPCE. The linear response was observed for concentration range of phospho-p5315 from 0.2 to 10 ng¨mL´1, with the detection limit of 0.1 ng¨mL´1.
## Vascular endothelial growth factor (vegf)
Vascular endothelial growth factor (VEGF) is an important growth factor that regulates the progression of angiogenesis. VEGF overexpression appears in many human cancers, such as brain tumors, lung cancer, breast cancer, gastrointestinal cancer and urinary tract tumors [bib_ref] The biology of vegf and its receptors, Ferrara [/bib_ref]. Lin et al. developed a simple and reusable strategy for detecting the VEGF concentration in human serum using a graphene based biosensor [bib_ref] A reusable magnetic graphene oxide-modified biosensor for vascular endothelial growth factor detection..., Lin [/bib_ref]. The sensing platform deposited at Au electrode surface consisted of a nanocomposite of GO-F 3 O 4 NPs functionalized with Avastin, as the specific biorecognition element for VEGF. The amperometric signal was measured by differential pulse voltammetry to quantify the VEGF concentration, being the linear response of the biosensensor observed for the range of VEGF concentrations from 0.03125 to 2 ng¨mL´1. The biosensors showed a good stability (RSD = 2.36%, n = 50) and a limit of detection of 0.03125 ng¨mL´1.
## Alpha-fetoprotein (afp)
Alpha-fetoprotein (AFP) is a glycoprotein of fetal component produced during the embryonic period. However, high AFP serum levels can be found mainly in patients with hepatocellular carcinoma or other types of cancer such as, lung cancer, biliary cancer, gastric cancer, pancreatic cancer, teratocarcinoma of the testis. Graphene-based materials have been explored as a sensing platform for the development of noble electrochemical immunoassay in order to amplify the sensitivity on detection of AFP. The pioneer work reported a graphene-based ultrasensitive multiplexed electrochemiluminescent (ECL) immunoassay method for the detection AFP tumor marker [bib_ref] Sandwich-type electrochemiluminescence immunosensor based on pdda-g@lu-au composite for alpha-fetoprotein detection, Gan [/bib_ref]. The graphene probe was synthesized by the assembly with poly (diallyldimethylammonium chloride) (PDDA) and luminol-capped gold nanoparticles (PDDA-G@Lu-Au). The obtained PDDA-G@Lu-Au composite particles were used as substrate for antibody and HRP immobilization with high luminol capacity load efficiency. The second probe consisted on multilabel-antibody functionalized core-shell Fe 3 O 4 @Au composites (GMPs). The combination of AFP with the two labels results in sandwich-type immunocomplexe PDDA-G@Lu-Au~HRP-Ab2/AFP/Ab1~GMP. The magnetic sandwich-type immunocomplexes were immobilized on the working screen-printed carbon electrode (SPCEs) by applying a magnetic field. That approach avoids some adverse effects obtained through the surface functionalization of the electrodes. Under the optimized conditions, the ECL method shows a linear range of AFP detection from 0.002 to 20 ng¨mL´1 and a low detection limit of 0.2 pg¨mL´1. Recently Zhuo et al. proposed the development of a nobel electrochemiluminescent immunosensor based on a new enhancer of peroxydisulfate system for the detection of AFP [bib_ref] Supramolecular assembly of perylene derivatives on au functionalized graphene for sensitivity enhancement..., Zhuo [/bib_ref]. The sensor probe design was based on rGO/AuNPs nanocomposites functionalized by supramolecular assembly with arginine covalently bond to 3,4,9,10-perylenetetracarboxylic acid (PTC-Arg/Au@Gra). The PTC-Arg/Au@Gra bioconjugate was used as multifunctional nanocarriers for the absorption of the second antibody of alpha-fetoprotein (Ab2), which formed sandwich-type immunoassay format through association with AFP and the first antibody (Ab1) previously assembled at Au electrode surface. With PTC-Arg/Au@Gra as enhancer of peroxydisulfate system, the immunosensor exhibited a wide dynamic range of 0.001-10 ng¨mL´1 and detection of limit of 0.3 pg¨mL´1.
## Multiplex cancer tumor markers detection
Graphene is starting to being explored as an effective platform for electrochemical multiplex detection of actively targeted biomarkers, which has clinical relevance. Recently it was reported the development of a suspended single crystalline graphene (SCG) biosensor for multiplex lung cancer tumor markers detection [bib_ref] Towards intrinsic graphene biosensor: A label-free, suspended single crystalline graphene sensor for..., Li [/bib_ref]. After the immobilization process of the antibodies, the label-free suspended SCG sensor was ready for detection of the important lung cancer biomarkers: ANXA2, VEGF, and ENO1. SCG label-free lung cancer biosensor showed a resistive response shift due to the absorption of biomolecules by the bio-receptors on the surface of graphene that cause the change in density and mobility of charge carriers of the film according to different concentrations of the three types of lung cancer tumor markers, ANXA2, VEGF, and ENO1. The biosensor sensitivity studies performed with presence of the three tumor markers showed that the response to the specific antigen is 1 order of magnitude larger than the non-specific ones. The high performance of the SCG biosensor results from synergistic effects obtained from the combination of two important bioevents on its surface: strong interaction between antigen and its bio-receptors at graphene surface and the blocking of the non-specific binding sites on graphene surface by BSA, which further avoids the absorption of non-specific molecules. Furthermore, the suspended structure of SCG biosensor, which has better carrier mobility, sensing area, and low frequency noise is responsible for the enhanced sensitivity with detection limit of 0.1 pg¨mL´1, good specificity and large linear detection range from 1 pg¨mL´1 to 1 µg¨mL´1. This SCG label-free lung cancer biosensor showed to have the lower detection limits ever reported for the three different tumors markers ANXA2, VEGF, and ENO1.
## Dna biosensors
DNA sensors are common used approach for early cancer diagnosis and mutation detection. Electrochemical sensors are one of most used approaches on DNA technology development, because DNA bases are electroactive which allows direct electrochemical signals recording during the electrooxidation process. Graphene demonstrate superior electrochemical performance for oxidation of DNA bases due to its outstanding electrochemical properties and large surface-to-volume ratio, that play a very important role on the development of new electrodes with increased sensitivity and simultaneous detection [bib_ref] Stacked graphene nanofibers for electrochemical oxidation of dna bases, Ambrosi [/bib_ref]. Electrochemical DNA graphene based sensors for low-concentration detection of Breast Cancer 1 (BRCA1) DNA sequences are a very recent technology [bib_ref] Graphene-dna electrochemical sensor for the sensitive detection of brca1 gene, Rasheed [/bib_ref]. It was reported that mutations on this gene were associated with an 80% increased risk of breast and ovarian cancer [bib_ref] Identifying and testing for hereditary susceptibility to common cancers. Ca-a, Sifri [/bib_ref]. The biosensors construct was based on the functionalization of GCE surface with rGO as a sensing platform for the immobilization of DNA capture probe (DNA-c with amino group at 5 1 end). In this system, one-half of the DNA target (DNA-t) is allowed to hybridize with the immobilized capture probe (DNA-c), while the other half interacts with the reporter probe (DNA-r) conjugated with gold nanoparticles. Electrochemical oxidation of gold nanoparticle was monitored using cyclic voltammetry to detect the concentration of DNA-t. The results showed that the sensor was stable, reproducible and sensitive and it could detect up to 6 fg¨mL´1 of DNA target. In another work, Wang et al. proposed a novel sensitive and selective electrochemical DNA graphene-based sensor for the detection of BCR/ABL fusion gene in chronic myelogenous leukemia (CML) [bib_ref] Graphene sheets, polyaniline and aunps based dna sensor for electrochemical determination of..., Wang [/bib_ref]. CML is a clonal neoplastic disorder of hematopoietic stem cells caused by expression of the chimeric BCR/ABL fusion oncogene (abnormality occurs in more than 95% patients) [bib_ref] Chronic myelogenous leukemia: Recent advances, Champlin [/bib_ref]. The proposed sensor structure consist on the surface functionalization of GCE trough the self-assembly of graphene/chitosan nanocomposite, followed by the electro-polymerization of polyaniline (PANI) and electrodeposition of Au nanoparticles and finally decorated with a functional hairpin structure probe for the detection of BCR/ABL fusion gene of CML. The hybridization event was sensitively transduced to the enzymatically amplified electrochemical current signals, showing a high DNA detection sensitivity and a low detection limit 2.11 pM.
## Cell sensors
Graphene based sensors were also explored for modern diagnosis of cancer by the detection and quantification of specific cancer cells. Graphene based electrochemical sensors has been revealed as the most promising method. Graphene based materials on cells sensors can provide an increase of electrochemical properties in the electrodes and furthermore provides very rich anchor sites to bind recognition species in order to create an interface with high selectivity. The pioneer work of Guo et al. reported a smart multilayer amperometric sensor based on deposition of rGO onto an ITO electrode, followed by the functionalization with Prussian blue as H 2 O 2 catalyst and laminin matrix protein to promote cell adhesion [fig_ref] Figure 6: SEM images of MCF-7 cells cultured [/fig_ref]. On this system it was observed that rGO can provide a compatible interface for the growth of human cells and excellent electrical conductivity for electrical detection, allowing for the first time, the in situ selective and quantitative extracellular H 2 O 2 detection. Breast cancer cells were grown on the electrode surface and the sensor was able to detect H 2 O 2 with a LOD of 0.1 µM upon stimulation [bib_ref] Biointerface by cell growth on layered graphene-artificial peroxidase-protein nanostructure for in situ..., Guo [/bib_ref].
Recently scientists started to give attention to the development new graphene ultrasensitive sensors able to quantify cells that can growth directly on its sensing surface or that can specifically detect cancer cells from a very complex mixture. Wu et al. develop a high sensitive graphene based electrochemical sensor through the surface modification of GCE with: chitosan/electrochemically rGO film covalently modified with anti-EpCAM antibodies [bib_ref] Highly specific and ultrasensitive graphene-enhanced electrochemical detection of low-abundance tumor cells using..., Wu [/bib_ref]. This biointerface was then used to detect Hep3B cancer cells. A sandwich system was then generated using CdTe-and ZnSe coated silica nanoparticles that could easily serve as tracing tags to label antiEpCAM and anti-GPC3. Each biorecognition event yields a distinct voltammetric peak, which position and size reflects the corresponding identity and amount of the respective antigen. The combination of ultrasensitive and highly specific electrochemical and fluorescent immunosensing methods showed great potential for application on the detection of circulating tumor cells. The Immunosensor developed exhibited high sensitivity and specificity with excellent stability, reproducibility, and accuracy, with a LOD low then 10 cells¨mL´1. In another work was reported the preparation of a graphene electrode modified with a new conjugate of peptide nanotubes and folic acid for the selective detection of human cervical cancer cells (HeLa cells) over-expressing folate receptors. The increases number of HeLa cells immobilized at electrode surface promotes a formation of insulating layer inducing the reduction of the recorded intensity of the electrochemical signals. This electrode provides a new sensing alternative for cancer cells detection, with high simplicity related with the synthetic process when compared with the previous one, however it showed higher LOD of 250 cells¨mL´1 [bib_ref] Detection of cancer cells using a peptide nanotube-folic acid modified graphene electrode, Castillo [/bib_ref]. Feng et al. develop a reusable electrochemical graphene-based sensor using aptamer clinical trial II AS1411 for label-free cancer cell detection [bib_ref] A graphene functionalized electrochemical aptasensor for selective label-free detection of cancer cells, Feng [/bib_ref]. Graphene surface was functionalized with 3,4,9,10-perylene tetracarboxylic acid (PTCA) through π-π stacking and hydrophobic interactions in order to introduce more -COOH groups. NH2-modified aptamer strand was linked to PTCA/rGO via covalent bond as the recognition element that can bind to the overexpressed nucleolin on the plasma membrane of cancer cells. The binding of cancer cells decreased the access of the redox probe to the electrode interface. The electrochemical aptasensor showed to be able to differentiate three types of cancer cells (HeLa cells (human cervical carcinoma cell), MDA-MB-231 (human breast cancer cell), K562 cells (leukemia line)) and normal ones (NIH3T3 cells). Another electrochemical graphene-based sensor was developed for specific detection of tumor marker Her2 overexpressed on surface of SKOV-3 tumor cells [bib_ref] Immunoassay for skov-3 human ovarian carcinoma cells using a graphene oxide-modified electrode, Xia [/bib_ref]. The GO modified electrode was covalently functionalized with Anti-Her2 for the capture of SKOV-3 cells, and the unbound HER2 was available to connect with the anti-HER2 graft dsDNA, integrating the advantages of graphene electrochemical properties and DNA marker. This approach provided a very low LOD 5.2 cells¨mL´1 for SKOV-3 cells.
work, Wang et al. proposed a novel sensitive and selective electrochemical DNA graphene-based sensor for the detection of BCR/ABL fusion gene in chronic myelogenous leukemia (CML) [bib_ref] Graphene sheets, polyaniline and aunps based dna sensor for electrochemical determination of..., Wang [/bib_ref]. CML is a clonal neoplastic disorder of hematopoietic stem cells caused by expression of the chimeric BCR/ABL fusion oncogene (abnormality occurs in more than 95% patients) [bib_ref] Chronic myelogenous leukemia: Recent advances, Champlin [/bib_ref]. The proposed sensor structure consist on the surface functionalization of GCE trough the self-assembly of graphene/chitosan nanocomposite, followed by the electro-polymerization of polyaniline (PANI) and electrodeposition of Au nanoparticles and finally decorated with a functional hairpin structure probe for the detection of BCR/ABL fusion gene of CML. The hybridization event was sensitively transduced to the enzymatically amplified electrochemical current signals, showing a high DNA detection sensitivity and a low detection limit 2.11 pM.
## Cell sensors
Graphene based sensors were also explored for modern diagnosis of cancer by the detection and quantification of specific cancer cells. Graphene based electrochemical sensors has been revealed as the most promising method. Graphene based materials on cells sensors can provide an increase of electrochemical properties in the electrodes and furthermore provides very rich anchor sites to bind recognition species in order to create an interface with high selectivity. The pioneer work of Guo et al. reported a smart multilayer amperometric sensor based on deposition of rGO onto an ITO electrode, followed by the functionalization with Prussian blue as H2O2 catalyst and laminin matrix protein to promote cell adhesion [fig_ref] Figure 6: SEM images of MCF-7 cells cultured [/fig_ref]. On this system it was observed that rGO can provide a compatible interface for the growth of human cells and excellent electrical conductivity for electrical detection, allowing for the first time, the in situ selective and quantitative extracellular H2O2 detection. Breast cancer cells were grown on the electrode surface and the sensor was able to detect H2O2 with a LOD of 0.1 µM upon stimulation [bib_ref] Biointerface by cell growth on layered graphene-artificial peroxidase-protein nanostructure for in situ..., Guo [/bib_ref]. [bib_ref] Low-frequency 1/f noise in graphene devices, Balandin [/bib_ref] -laminin (only one layer of laminin on top of the structure), and 5. ITO/(graphene-AP-laminin) [bib_ref] Low-frequency 1/f noise in graphene devices, Balandin [/bib_ref]. Reproduced with permission [bib_ref] Biointerface by cell growth on layered graphene-artificial peroxidase-protein nanostructure for in situ..., Guo [/bib_ref]. Copyright 2010, WILEY-VCH Verlag GmbH & Co.
Recently, graphene/inorganic nanoparticles hybrids started to be used as a sensing platform at electrodes surface in order to increase the sensitivity of the sensor. Liu et al. developed a new ZnO/graphene composite modified with S6 aptamer as sensitive photoelectrochemical (PEC) strategy for the specific detection of SK-BR-3 cancer cells [bib_ref] Application of zno/graphene and s6 aptamers for sensitive photoelectrochemical detection of sk-br-3..., Liu [/bib_ref]. This hybrid structure was used as sensing platform on ITO working electrode, in order improve its PEC performance. The increase of photocurrent intensity results from the increase in steric hindrances with S6 aptarmer with SK-BR-3 cancer cells receptors. The results showed high selectivity with analogous cells and a LOD of 58 cells¨mL´1. Yan et al. reported a novel graphene immunosensor for detection of cancer cells MCF-7 though the surface modification of the working electrode with GO/AuNPs nanocomposite [bib_ref] An aptasensor for sensitive detection of human breast cancer cells by using..., Yan [/bib_ref]. The composite was functionalized with aptamer to recognize and bind the tumor marker on the surface of cancer cells MCF-7. Furthermore, thionine functionalized nanoporous PtFe alloy was used as signal amplifier for cells detection. The results obtained showed the increase of the electrochemical signal measured proportional to the concentration of MCF-7 cells. The biosensor showed a good selectivity, acceptable stability and reproducibility with a LOD of 38 cell¨mL´1. Another work reported the use of small bifunctional composite quantum dot (Fe 3 O 4 /CdSe QD), with intense electrochemiluminescence (ECL) and excellent magnetic property, as reported as enhancer signal probe for sensitive detection of cancer cells via DNA cyclic amplification technique using GO sensing platform to immobilize DNA capture probes (c-DNA1) at the surface of Au electrode [bib_ref] Amplified electrochemiluminescence detection of cancer cells using a new bifunctional quantum dot..., Jie [/bib_ref]. The increased concentration of target cells results in scission of more QDs/DNA signal probe, thus more decrease of ECL signal was obtained. The relationship between the changes of ECL signal and the concentrations of target cells was for the range from 300 to 9000 cells¨mL´1 with a detection limit of 98 cells¨mL´1.
## Luminescence sensors
Graphene as we discuss before was characterized to process very interesting properties, in particular high conductivity and luminescence quenching ability. In fact, the ultra-high quenching efficiency is attributed to three possible mechanisms: Forter resonance energy transfer (FRET), surface energy transfer (SET) and photo-induced electron transfer [bib_ref] Mid-infrared plasmonic biosensing with graphene, Rodrigo [/bib_ref]. As a quencher for diverse luminescence energy donors, graphene materials have been used in combination with several probes.
Graphene oxide (GO) based fluorescent biosensor was prepared for real-time in situ detection of integrin αvβ3. Integrin have a vital role in cancer cell adhesion, proliferation, migration and metastasis [bib_ref] Why integrin as a primary target for imaging and therapy, Niu [/bib_ref]. The biosensor concept is based on the capacity of the sp 2 aromatic domains of GO to establish noncovalent interactions with pyrene molecules resulting in the quenching effect via fluorescence resonance energy transfer or dipole-dipole coupling effects. On this work GO based biosensor system was initially at a quenching state due to the proximity of RGD-pyrene to GO upon π-π stacking interactions [bib_ref] A nanoscale graphene oxide-peptide biosensor for real-time specific biomarker detection on the..., Wang [/bib_ref]. However, the competitive binding of an RGD receptor, integrin avb3, to the RGD ligand disturbs the adsorption of RGD-pyrene onto the GO surface, resulting in the recovery of pyrene fluorescence with the increasing of concentrations of integrin as illustrated in [fig_ref] Figure 7: Real-time in situ detection of breast cancer cell surface integrin expression by... [/fig_ref]. The biosensors developed showed the capacity for detection of purified integrin protein in buffer and the effectiveness for in situ detection of integrin overexpression on cancer cells surface (MDA-MB-435 cell line).
A multiplex microfluidic chip integrated with the GO-based FRET strategy to create a screening assay for in situ detection of tumor cells was also reported [bib_ref] Visual and high-throughput detection of cancer cells using a graphene oxide-based fret..., Cao [/bib_ref]. For this purpose, a 33-channel microfluidic chip integrated with the GO-based FRET aptasensor was designed and employed to detect target CCRF-CEM cells with a confocal fluorescence scanning microscope. Initially the FRET probe of GO/FAM-Sgc8 exhibited a quenched fluorescence because of π-π stacking interactions between FAM-Sgc8 and GO. The strong interaction between FAM-Sgc8 and CCRF-CEM cells promotes the release FAM-Sgc8 from GO surface resulting in the recovery of the FAM-Sgc8 fluorescence. The changes observed in fluorescence intensity measurement allowed the quantification of the target cancer cells CCRF-CEM, with the linear response in a concentration range from 2.5ˆ10 1 to 2.5ˆ10 4 cells¨mL´1 and a detection limit about 25 cells¨mL´1. Another work suggested a new graphene based fluorescent biosensor system based on a development of a novel molecular aptamer beacon (MA) as fluorescently labelled and GO as the acceptor for target detection by employing long range resonance energy transfer (LrRET) of cellular prion protein (PrPC) [bib_ref] Sensitive detection of prion protein through long range resonance energy transfer between..., Zhuang [/bib_ref]. Initially the fluorescence of the designed MAB was completely quenched by GO, however after addition of PrPC the quenched fluorescence was recovered significantly. The results obtained showed that PrPC can be detected over a wide range of 10.2-78.8 µg¨mL´1 with a detection limit of 0.309 µg¨mL´1.
π-π stacking interactions [bib_ref] A nanoscale graphene oxide-peptide biosensor for real-time specific biomarker detection on the..., Wang [/bib_ref]. However, the competitive binding of an RGD receptor, integrin avb3, to the RGD ligand disturbs the adsorption of RGD-pyrene onto the GO surface, resulting in the recovery of pyrene fluorescence with the increasing of concentrations of integrin as illustrated in [fig_ref] Figure 7: Real-time in situ detection of breast cancer cell surface integrin expression by... [/fig_ref]. The biosensors developed showed the capacity for detection of purified integrin protein in buffer and the effectiveness for in situ detection of integrin overexpression on cancer cells surface (MDA-MB-435 cell line). A multiplex microfluidic chip integrated with the GO-based FRET strategy to create a screening assay for in situ detection of tumor cells was also reported [bib_ref] Visual and high-throughput detection of cancer cells using a graphene oxide-based fret..., Cao [/bib_ref]. For this purpose, a 33-channel microfluidic chip integrated with the GO-based FRET aptasensor was designed and employed to detect target CCRF-CEM cells with a confocal fluorescence scanning microscope. Initially the FRET probe of GO/FAM-Sgc8 exhibited a quenched fluorescence because of π-π stacking interactions between FAM-Sgc8 and GO. The strong interaction between FAM-Sgc8 and CCRF-CEM cells promotes the release FAM-Sgc8 from GO surface resulting in the recovery of the FAM-Sgc8 fluorescence. The changes observed in fluorescence intensity measurement allowed the quantification of the target cancer cells CCRF-CEM, with the linear response in a concentration range from 2.5 × 10 1 to 2.5 × 10 4 cells mL -1 and a detection limit about 25 cells·mL −1 . Another work suggested a new graphene based fluorescent biosensor system based on a development of a novel molecular aptamer beacon (MA) as fluorescently labelled and GO as the acceptor for target detection by employing long range resonance energy transfer (LrRET) of cellular prion protein (PrPC) [bib_ref] Sensitive detection of prion protein through long range resonance energy transfer between..., Zhuang [/bib_ref]. Initially the fluorescence of the designed MAB was completely quenched by GO, however after addition of PrPC the quenched fluorescence was recovered significantly. The results obtained showed that PrPC can be detected over a wide range of 10.2-78.8 µg·mL −1 with a detection limit of 0.309 µg·mL −1 .
# Conclusions/outlook
To summarize, we have highlighted the potential of graphene and its derivatives for detection and quantification of cancer biomolecules and cells. Furthermore, we also described numerous procedures capable of convert graphene via surface engineering into specific platforms for particular types of biomolecules or bioreceptors overexpressed by cancerous cells. The biosensing of cancer was discussed giving light to the synergistic properties of the graphene-based sensing entities that can simultaneously act as imaging and therapeutic agents. Additionally, the bioquantification of cancer biomarkers and cells was fully described giving particular attention to specific biomarkers
# Conclusions/outlook
To summarize, we have highlighted the potential of graphene and its derivatives for detection and quantification of cancer biomolecules and cells. Furthermore, we also described numerous procedures capable of convert graphene via surface engineering into specific platforms for particular types of biomolecules or bioreceptors overexpressed by cancerous cells. The biosensing of cancer was discussed giving light to the synergistic properties of the graphene-based sensing entities that can simultaneously act as imaging and therapeutic agents. Additionally, the bioquantification of cancer biomarkers and cells was fully described giving particular attention to specific biomarkers responsible for well-known cancer types.
Although the great potentialities of graphene in biosensing technologies, its use could present some health concern issues since a large number of toxicological studies presented contradictory conclusions . In vitro and in vivo studies have reported that the cytotoxicity of graphene and its derivatives depend on many factors, including preparation protocol, purification processes and final physicochemical properties [bib_ref] Nano-graphene oxide: A potential multifunctional platform for cancer therapy, Gonçalves [/bib_ref]. Thus, there is a long way to go until these graphene based sensing-systems become optimize and subsequently available for real-world clinical applications. In fact, more detailed studies concerning, for example, the safety of graphene and its derivatives should be addressed with the purpose of monitoring and controlling their metabolic pathway, cellular-uptake mechanism and long term toxicity, which are key-points for nanomaterials applications in bioimaging, drug delivery and cancer therapy.
The scalable, controllable and reproducible methods of synthesis of graphene-based nanomaterials are other relevant challenges that need to be improved in order to obtain consistent results.
Overall, cancer is a disease with high innate heterogeneity, very difficult to detect and diagnose via a single biomarker with high specificity and sensitivity. The detection of cancer biomarker in real biological samples such as blood plasma, blood serum cerebrospinal fluid, saliva or urine is a real challenge since these samples are very complex systems with different kinds of proteins, ions and other chemical species able to promote false positive responses in label free assays. Possible ways to overcome this issue are the passivation of the sensor's surface with an antifouling agent or, more particularly, the previous filtration/purification of the biological sample in order to concentrate the target entity.
Thus, as it was discussed, the ability of graphene based materials to conjugate their outstanding chemical and physical features into remarkable sensing properties appears to be a solid milestone on the long pathway to achieve an early and optimal cancer diagnostics. Indeed, researchers around the world are continuing to explore the wide range of sensing strategies opened by graphene in the last few years due to its proven adaptability and functionality in different cancer microenvironments. For the near future, the ultimate goal is to develop graphene based devices capable of simultaneously detect multiple cancer biomarkers.
[fig] Figure 1: Illustration of graphene derivatives with potential applications on cancer biosensors. [/fig]
[fig] Figure 2: Fluorescent images of human breast cancer cell T47D after incubation with green GQDs for 4 h (a) phase contrast picture of T47D cells; (b) Individual nucleus stained blue with DAPI; (c) Agglomerated green GQDs surrounding each nucleus; (d) The overlay high contrast image of nucleolus stained with blue DAPI and GQDs (green) staining. Reproduced with permission [61] Copyright 2012, American Chemical Society. [/fig]
[fig] Figure 3: (a) In vivo fluorescence images of GQD-HA in mice after tail vein injection; (b) Ex vivo images of liver, kidney, spleen, heart, and tumor after dissection; (c) Normalized intensity from dissected organs .Reprinted with permission from[58]. Copyright (2013) American Chemical Society. [/fig]
[fig] Figure 4: The cellular uptake mechanism investigations of GO and Au/GO hybrids. Hela 229 cells are incubated for 2 h with GO (a-d) or Au/GO hybrids (e-h) at 37 °C (a, b, e and f) and 4 °C (c, d, g and h), respectively. The left panels are optical images while the right panels are the corresponding Raman images (scale bar: 5 mm). Reproduced with permission[80]. Copyright 2012, Royal Society of Chemistry. [/fig]
[fig] Figure 5: Schematic Illustration of the Multi-enzyme Labeling Amplification Strategy Using HRP-p53392Ab2-GO Conjugate. Reproduced with permission[110]. Copyright 2011, American Chemical Society. [/fig]
[fig] Figure 6: SEM images of MCF-7 cells cultured (12 h) on (a) ITO/(AP)10 and (b) ITO/(graphene-AP)10. The scale bar is 5 µm for (a) and (b). (c) Proliferation curves of cells cultured on fi lms with different compositions: 1. ITO/(AP)10, 2. ITO/(graphene)10, 3. ITO/(graphene-AP)10, 4. ITO/(graphene-AP)10-laminin (only one layer of laminin on top of the structure), and 5. ITO/(graphene-APlaminin)10. Reproduced with permission [120]. Copyright 2010, WILEY-VCH Verlag GmbH & Co. [/fig]
[fig] Figure 7: Real-time in situ detection of breast cancer cell surface integrin expression by the RGDpyrene-GO probe: (a) probe fluorescence recovery by live MDA-MB-435 cancer cells which overexpress integrin avb3 on the cell surface. The recovered fluorescence is mainly detected on the cell membrane as indicated by white arrows; (b) probe fluorescence recovery by MDA-MB-435 cancer cells followed by 4% formalin fixing; (c) equivalent concentration of free RGD-pyrene incubated with liveMDA-MB-435 demonstrates significant endocytosis as indicated by white arrows; (d) equivalent concentration of RGD-pyrene incubated with MDA-MB-435 followed by 4% formalin fixing. Reproduced with permission [129]. Copyright 2012, Royal Society of Chemistry. [/fig]
[fig] Figure 8: (a) Schematic setup and (b) the photograph of the GO-based FRET aptasensing microfluidic chip. Scale bar: 10 mm. (c) The principle of a 'signal-on' aptasensor for detecting CCRF-CEM cells by assaying the cell-induced fluorescence recovery of GO/FAM-Sgc8. Reproduced with permission [130] Copyright 2012, Royal Society of Chemistry. [/fig]
[table] Table 1: Sandwich-type graphene-based biosensor performance for the detection of carcinoembryonic antigen (CEA). [/table]
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Fasciclin-like arabinogalactan proteins, PtFLAs, play important roles in GA-mediated tension wood formation in Populus
In Populus, the transcripts of fasciclin-like arabinogalactan proteins (FLAs) are accumulated in tension wood (TW) xylem, however their biological functions in TW formation are largely unknown. In this work, we demonstrated that PtFLA6, one of poplar TW-associated PtFLAs, was abundantly expressed in TW, and mainly localized in differentiating G-fibers. The bended stems of PtFLA6 antisense transgenic poplar showed decreased transcripts of PtFLAs, including PtFLA6, and reduced PtFLA6 like proteins, leading to inhibited TW differentiation and formation. We also showed that gibberellin A3 (GA 3 ) was enriched in the xylem of TW side, accompanied with a lowered level of PtRGA1, a poplar DELLA protein. When GA 3 biosynthesis was restrained in the bended poplar stems by a GA biosynthesis inhibitor (daminozide), TW formation was obviously repressed, as a result of restricted PtRGA1 degradation, and reduced PtFLA6 like proteins and PtFLA expression. Further studies indicated that PtFLAs were negatively regulated by PtRGA1. This study suggests that PtFLAs play important roles in the poplar TW formation, possibly regulated by GA signaling.
As perennial plants, trees are often inclined or bended when they encounter gravitation or mechanical stresses such as wind, snow, and growth on slope. In response to the perception of gravity or mechanical stimuli, woody plants form an abnormal tissue, named reaction wood (RW). RW, formed on the upper side of leaning or bended stems in angiosperms, is called tension wood (TW) [bib_ref] The chemical composition of tension wood, Timell [/bib_ref] , which generates a tensile force in order to pull the displaced stem to its original position 2 .
TW is characterized by a higher proportion of fibers and a lower proportion of vessel elements [bib_ref] Anatomical characteristics of tension wood and opposite wood in young inclined stems..., Jourez [/bib_ref]. The most striking modification is the presence of an additional thick gelatinous cell wall layer (G-layer) in the TW fibers. The G-layer is a layer of the secondary wall (replacing most of the S2 and the entire S3 layer) and is mainly composed of crystalline cellulose [bib_ref] The chemical composition of tension wood, Timell [/bib_ref] [bib_ref] Effect of gravitational stimulus duration on tension wood formation in young stems..., Jourez [/bib_ref]. Although TW is considered to reduce the wood quality and be a nuisance in wood processing and paper industry, it is beneficial to increase the glucose yields in biofuel industry [bib_ref] Reaction wood-a key cause of variation in cell wall recalcitrance in willow, Brereton [/bib_ref]. Therefore, study on the molecular mechanism of TW formation is becoming more scientifically and commercially meaningful.
Tension wood formation has been thought to be regulated by plant hormones, such as auxin, ethylene and gibberellins (GAs). During TW formation, expressions of several Aux/IAA genes were changed [bib_ref] Environmental and auxin regulation of wood formation involves members of the Aux/IAA..., Moyle [/bib_ref] and the auxin transporter PIN3 transported auxin toward the cambium in the TW side to trigger TW formation [bib_ref] Transcriptional and hormonal regulation of gravitropism of woody stems in Populus, Gerttula [/bib_ref]. However the balance of endogenous auxin level was not significantly altered [bib_ref] Patterns of auxin distribution during gravitational induction of reaction wood in poplar..., Hellgren [/bib_ref] , which indicated that auxin may not directly regulate TW formation. Ethylene has been confirmed as a key regulator in poplar TW formation. In the gravity-induced tension wood tissues of Populus, both the expression of PttACO1 which encodes a 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase in ethylene biosynthesis pathway, and the enzyme activity of ACC were increased [bib_ref] Asymmetric expression of a poplar ACC oxidase controls ethylene production during gravitational..., Andersson-Gunnerås [/bib_ref]. On the other hand, in ethylene insensitive transgenic trees and those treated with ethylene perception inhibitor [1-methylcyclopropene (1-MCP)], TW formation was obviously repressed [bib_ref] Ethylene is an endogenous stimulator of cell division in the cambial meristem..., Love [/bib_ref]. GA is another kind of plant hormones regulating tension wood formation. Application of GA could induce TW formation in angiosperm trees [bib_ref] Gibberellin-induced formation of tension wood in angiosperm trees, Funada [/bib_ref]. A recent report suggested that GA controlled auxin transport during TW formation, and that GA stimulated TW formation was sensitive to ARK2 levels [bib_ref] Transcriptional and hormonal regulation of gravitropism of woody stems in Populus, Gerttula [/bib_ref]. However the exact regulatory mechanism of GA-mediated TW formation is still unclear.
FLAs (fasciclin-like AGPs), containing one or more fasciclin domain(s) [bib_ref] The complex structures of arabinogalactan-proteins and the journey towards understanding function, Gaspar [/bib_ref] , belong to the arabinogalactan proteins (AGPs), and are subdivided into four groups named A to D [bib_ref] The fasciclin-like arabinogalactan proteins of Arabidopsis. A multigene family of putative cell..., Johnson [/bib_ref]. FLAs played important roles in seed coat mucilage, normal cell expansion, stem biomechanics and cell wall architecture in Arabidopsis, and fiber initiation and elongation in cotton [bib_ref] The Arabidopsis SOS5 locus encodes a putative cell surface adhesion protein and..., Shi [/bib_ref] [bib_ref] Fasciclin-like arabinogalactan proteins: specialization for stem biomechanics and cell wall architecture in..., Macmillan [/bib_ref] [bib_ref] Cellulose synthesis via the FEI2 RLK/SOS5 pathway and cellulose synthase 5 is..., Harpaz-Saad [/bib_ref] [bib_ref] A Fasciclin-Like Arabinogalactan Protein, GhFLA1, Is Involved in Fiber Initiation and Elongation..., Huang [/bib_ref]. Interestingly, a group of FLAs belonging to subgroup A were highly up-regulated in the TW tissues compared with that in the opposite wood (OW) tissues of poplar [bib_ref] Transcriptional and hormonal regulation of gravitropism of woody stems in Populus, Gerttula [/bib_ref] [bib_ref] Poplar genes encoding fasciclin-like arabinogalactan proteins are highly expressed in tension wood, Lafarguette [/bib_ref] [bib_ref] Biosynthesis of cellulose-enriched tension wood in Populus: global analysis of transcripts and..., Andersson-Gunnerås [/bib_ref] [bib_ref] Transcriptome profiling the basal region of poplar stems during the early gravitropic..., Azri [/bib_ref]. In addition, a willow FLA gene SxFLA12 was also specifically expressed in developing TW [bib_ref] Transcriptional and hormonal regulation of gravitropism of woody stems in Populus, Gerttula [/bib_ref]. All these reports indicate that FLAs may play important roles during TW formation of woody plants. However, the biological functions of FLAs in TW formation are not well illustrated yet.
In a previous study, we found that PtFLAs affected the poplar stem biomechanics by altering cell wall compositions in Populus 21 . In the present work, we further investigated the biological function of PtFLAs in mechanical stress induced TW formation using the bended PtFLA6 antisense transgenic poplar plants. Our results suggest that PtFLAs play important roles in the TW formation of poplar.
# Results
PtFLA6 is specifically expressed in TW. TW formation can be induced by gravitational stimulus [bib_ref] Anatomical characteristics of tension wood and opposite wood in young inclined stems..., Jourez [/bib_ref] [bib_ref] Effect of gravitational stimulus duration on tension wood formation in young stems..., Jourez [/bib_ref] , as well as by mechanical stress [bib_ref] Transcript profiling of Eucalyptus xylem genes during tension wood formation, Paux [/bib_ref]. In this study, we bended the stems of poplar plants grown in greenhouse to induce TW formation [fig_ref] Figure 1: PtFLA6 expression analyses during TW formation [/fig_ref]. As expected, obvious TW was produced in the upper side of the bended stems and stained into blue color by safranin/astra blue double staining; and OW in the lower side remained in red color [fig_ref] Figure 1: PtFLA6 expression analyses during TW formation [/fig_ref].
It has been reported that a group of FLA genes in poplar (PopFLA1-10, also named PtFLA12o, -K, -i, -Q, -S, -D, -V, -u, and -G) were highly up-regulated in tension wood [bib_ref] Poplar genes encoding fasciclin-like arabinogalactan proteins are highly expressed in tension wood, Lafarguette [/bib_ref] [bib_ref] Biosynthesis of cellulose-enriched tension wood in Populus: global analysis of transcripts and..., Andersson-Gunnerås [/bib_ref] , implying their important but unclear functions in TW formation. Based on the nucleotide sequences of PopFLA1-10, we extracted ten FLAs from the Joint Genome Initiative poplar database (http://genome.jgi.doe.gov/Poptr1_1/Poptr1_1.home.html) and named them as PtFLA1-10, respectively. All PtFLAs share very high similarity, and PtFLA2 and PtFLA3 have exactly the same nucleotide sequence [bib_ref] Antisense expression of the fasciclin-like arabinogalactan protein PtFLA6 gene in Populus inhibits..., Wang [/bib_ref]. We then confirmed the expression pattern of PtFLAs during TW formation by qRT-PCR analysis [fig_ref] Figure 1: PtFLA6 expression analyses during TW formation [/fig_ref]. The results showed that all PtFLAs were specifically expressed in the xylem tissues of vertically grown stems under normal growth condition in the greenhouse. When the stems were bended to induce TW fromation, PtFLAs were also mainly expressed in the xylem tissues in both TW and OW sides. However, all PtFLAs were highly expressed in the xylem tissues of the TW side compared to that of the OW side or the xylem tissues of normally grown stems. These results indicate that PtFLAs are up-regulated during TW formation to play some particular roles.
To understand the roles of PtFLAs in mechanical stress induced TW formation, we selected PtFLA6 with the most specific and abundant expression in TW for further study [bib_ref] Poplar genes encoding fasciclin-like arabinogalactan proteins are highly expressed in tension wood, Lafarguette [/bib_ref] [bib_ref] Biosynthesis of cellulose-enriched tension wood in Populus: global analysis of transcripts and..., Andersson-Gunnerås [/bib_ref]. We firstly confirmed the expression pattern of PtFLA6 during TW formation by Western blotting analysis. PtFLA6 proteins were predominantly accumulated in the xylem tissues of the TW side compared to that of the OW side [fig_ref] Figure 1: PtFLA6 expression analyses during TW formation [/fig_ref] , in consistence with its gene expression pattern [fig_ref] Figure 1: PtFLA6 expression analyses during TW formation [/fig_ref]. To further detect the detailed expression of PtFLA6 during TW formation, we then carried out promoter-GUS analysis. The promoter region (~1.5 kb) of PtFLA6 was cloned and used to construct the ProPtFLA6::GUS vector for poplar transformation. Two-month-old transgenic plants grown in greenhouse were bended for two weeks to induce TW formation. GUS staining showed that PtFLA6 promoter was mainly activated in the TW tissues compared with the phloem and OW tissues [fig_ref] Figure 1: PtFLA6 expression analyses during TW formation [/fig_ref]. These results reveal that PtFLA6 is a classical TW-associated protein.
PtFLA6 proteins are predominantly accumulated in differentiating G-fiber cells. Proteins only play functions in particular tissues and cells. To observe the precise tissue and cellular locations of PtFLA6 proteins during TW formation, immunohistochemical localization using PtFLA6 antibodies was carried out. Compared with the phloem and OW tissues, high density of green-brown signals representing PtFLA6 proteins were detected in the TW tissues [fig_ref] Figure 2: Immunohistochemical analysis of PtFLA6 during TW formation [/fig_ref] , whereas no staining was observed in the control samples [fig_ref] Figure 2: Immunohistochemical analysis of PtFLA6 during TW formation [/fig_ref]. Further analyses revealed that PtFLA6 proteins were abundantly accumulated in the differentiating G-fibers (DGFs) undergoing G-layer formation, and in the mature G-fibers (MGFs) with obvious G-layer [fig_ref] Figure 2: Immunohistochemical analysis of PtFLA6 during TW formation [/fig_ref]. This observation was more distinct under higher magnification of the images. PtFLA6 signaling was weaker in the radial expansion zone (REZ) [fig_ref] Figure 2: Immunohistochemical analysis of PtFLA6 during TW formation [/fig_ref] , but became stronger in the cell wall of DGF [fig_ref] Figure 2: Immunohistochemical analysis of PtFLA6 during TW formation [/fig_ref] , and gradually reduced as the G-fibers matured [fig_ref] Figure 2: Immunohistochemical analysis of PtFLA6 during TW formation [/fig_ref]. In addition, PtFLA6 proteins were mainly present in the outer wall of MGF other than in the G-layer [fig_ref] Figure 2: Immunohistochemical analysis of PtFLA6 during TW formation [/fig_ref].
To confirm the location of PtFLA6 proteins in the cell wall of GF (G-fiber), immunogold labeling TEM analysis was performed. During TW formation, G-layer began to form in the DGF [fig_ref] Figure 3: Immunocytolocalization of PtFLA6 in the G-fiber cells of TW xylem tissues [/fig_ref] which then developed into mature GF containing three distinct layers: primary wall (PW) with high electron opacity, secondary wall (SW) and thick G-layer with low electron opacity [fig_ref] Figure 3: Immunocytolocalization of PtFLA6 in the G-fiber cells of TW xylem tissues [/fig_ref]. The immunogold signals of PtFLA6, shown as black dots, were abundant in cell wall of DGF as well as in cytoplasm [fig_ref] Figure 3: Immunocytolocalization of PtFLA6 in the G-fiber cells of TW xylem tissues [/fig_ref]. However, the signals significantly decreased in MGF and mainly presented in PW and SW compared with the G-layer [fig_ref] Figure 3: Immunocytolocalization of PtFLA6 in the G-fiber cells of TW xylem tissues [/fig_ref]. The signals were more obvious under higher magnification of the images [fig_ref] Figure 3: Immunocytolocalization of PtFLA6 in the G-fiber cells of TW xylem tissues [/fig_ref]. In the negative control, no signal was observed [fig_ref] Figure 3: Immunocytolocalization of PtFLA6 in the G-fiber cells of TW xylem tissues [/fig_ref]. All these results indicate that PtFLA6, as a TW-associated protein, may mainly function in the G-fiber differentiation during TW formation.
Antisense expression of PtFLA6 inhibits TW formation. To illustrate the role of PtFLAs in TW formation, two-month-old PtFLA6 antisense transgenic plants (lines 4 and 14) grown in greenhouse were bended for ten days to induce TW formation. Then, the xylem tissues in the TW side were collected for qRT-PCR analysis. Expressions of most PtFLAs (PtFLA1-8) were down-regulated and reduced by 40% to 80% in the TW xylem of transgenic poplar plants compared with that in the TW xylem of wild type (WT) [fig_ref] Figure 4: TW formation was inhibited in PtFLA6 antisense transgenic plants [/fig_ref] , indicating that antisense silencing of PtFLA6 could also reduce the transcriptions of PtFLAs. Consistently, PtFLA6 proteins were obviously decreased in the TW xylem tissues of transgenic plants [fig_ref] Figure 4: TW formation was inhibited in PtFLA6 antisense transgenic plants [/fig_ref]. By histochemical staining, we observed that TW formation in the upper side of bended transgenic stems was inhibited, leading to a lowered TW-to-xylem area ratio [fig_ref] Figure 4: TW formation was inhibited in PtFLA6 antisense transgenic plants [/fig_ref]. We further investigated the G-fibers in the defined position of TW tissues by TEM analysis. The results showed that most mature G-fibers of WT plants contained a thick G-layer. However, more abnormal G-fibers (AGFs) with a thin G-layer were present in the TW tissues of transgenic plants [fig_ref] Figure 4: TW formation was inhibited in PtFLA6 antisense transgenic plants [/fig_ref]. In addition, the G-layer thickness of mature GF was also measured, and no significant difference was observed between WT and transgenic plants [fig_ref] Figure 2: Immunohistochemical analysis of PtFLA6 during TW formation [/fig_ref]. All these results reveal that PtFLAs play important roles in TW formation and differentiation. GA 3 biosynthesis and GA signaling are enhanced in TW xylem. To illuminate how PtFLAs were regulated during TW formation, we further analyzed the regulatory elements in their promoters. GA responsive elements were commonly found in the promoters of most PtFLAs (data not shown). More importantly, expressions of all PtFLAs (PtFLA1-10) were significantly induced by exogenous GA 3 treatment [fig_ref] Figure 5: Asymmetrical distribution of GA 3 during TW formation [/fig_ref] , indicating that GA 3 may be an upstream regulator of PtFLAs during TW formation. To demonstrate this supposition, we first confirmed whether GA 3 was a direct hormonal regulator involved in TW formation. By analyzing the gene expression pattern, we found that PttGA20ox1, a key endogenous GA biosynthesis gene [bib_ref] Tissue-specific localization of gibberellins and expression of gibberellin-biosynthetic and signaling genes in..., Israelsson [/bib_ref] , was highly up-regulated in the TW xylem compared with that in the OW xylem [fig_ref] Figure 5: Asymmetrical distribution of GA 3 during TW formation [/fig_ref]. Meanwhile, GA 3 content increased about two folds in the TW xylem compared to that in the OW xylem [fig_ref] Figure 5: Asymmetrical distribution of GA 3 during TW formation [/fig_ref]. This result was also confirmed by immunohistochemical test using GA 3 monoclonal antibody, showing that GA 3 signals in the TW tissues were obviously stronger than that in the OW tissues [fig_ref] Figure 3: Immunocytolocalization of PtFLA6 in the G-fiber cells of TW xylem tissues [/fig_ref].
Increased GA 3 content during TW formation may have triggered the GA signaling. In plants, GA signaling is mediated by DELLA proteins which function as the conserved repressors in GA-induced growth and development [bib_ref] Molecular mechanism of gibberellin signaling in plants, Sun [/bib_ref] [bib_ref] The molecular mechanism and evolution of the GA-GID1-DELLA signaling module in plants, Sun [/bib_ref]. Therefore, we investigated the protein content of the poplar DELLA proteins using AtRGA1 antibody. AtRGA1, as the key DELLA protein in Arabidopsis, is the homology of a putative Populus DELLA protein PtRGA1. Before performing the Western blotting, we examed the AtRGA1 antibody and found that it could successfully identify the purified PtRGA1 recombinant protein [fig_ref] Figure 4: TW formation was inhibited in PtFLA6 antisense transgenic plants [/fig_ref]. Western blotting analysis using bended stems of wild type plants demonstrated that PtRGA1 was obviously reduced in the TW xylem tissues compared with that in the OW xylem tissues, implying an enhanced GA signaling during TW formation [fig_ref] Figure 5: Asymmetrical distribution of GA 3 during TW formation [/fig_ref]. These results suggest that GA and GA signaling may play crucial roles in TW formation.
## Tw formation and ptfla expression are inhibited by a ga biosynthesis inhibitor.
To investigate the roles of GA 3 during TW formation, a GA biosynthesis inhibitor daminozide was used to treat the bended stems of wild type poplar. Histochemical staining analysis showed that TW formation was obviously repressed in the bended stems treated with daminozide, leading to a significantly reduced TW-to-xylem ratio [fig_ref] Figure 6: TW formation was inhibited by daminozide treatment [/fig_ref]. Then, the transcriptional level of PttGA20ox1 and the content of GA 3 were analyzed. We found that, accompanying a severe reduction in GA 3 content, PttGA20ox1 expression was significantly decreased in the TW side of bended stems treated with daminozide [fig_ref] Figure 6: TW formation was inhibited by daminozide treatment [/fig_ref]. In addition, Western blotting results showed that more PtRGA1 proteins were accumulated in the TW xylem of the daminozide treated stems than in that of the control [fig_ref] Figure 6: TW formation was inhibited by daminozide treatment [/fig_ref]. All these results demonstrate that GA plays an important role in regulating poplar TW formation.
Based on the result that PtFLAs were induced by exogenous GA 3 [fig_ref] Figure 5: Asymmetrical distribution of GA 3 during TW formation [/fig_ref] , we speculated that expressions of PtFLAs could be repressed by daminozide treatment. As we expected, PtFLA6 protein content was obviously decreased in TW xylem tissues of the daminozide treated stems [fig_ref] Figure 6: TW formation was inhibited by daminozide treatment [/fig_ref]. Meanwhile, the transcription levels of all PtFLA genes were decreased in the tissues of bended stems treated with daminozide [fig_ref] Figure 6: TW formation was inhibited by daminozide treatment [/fig_ref]. All these results indicate that GA can regulate the expression of PtFLAs during poplar TW formation.
PtRGA1 negatively regulates PtFLAs. To understand how the expression of PtFLAs is regulated by GA signaling, transient expression assays were performed. We found that the activity of PtFLA promoters was repressed by PtRGA1. When the effector of PtRGA1 was co-expressed with each reporter of PtFLA promoters (PtFLA1, PtFLA6 and PtFLA9) in tobacco leaves, the fluorescence signals were weaker than that of the control [fig_ref] Figure 7: PtRGA1 negatively regulates the expression of PtFLAs [/fig_ref]. In addition, by the transient expression of PtRGA1 in poplar xylem protoplasts, we found that transcriptional levels of all PtFLAs were obviously decreased in the transfected protoplasts overexpressing PtRGA1 [fig_ref] Figure 7: PtRGA1 negatively regulates the expression of PtFLAs [/fig_ref]. These results imply that the poplar DELLA protein PtRGA1 negatively regulates the expression of PtFLAs.
# Discussion
When TW was produced in the upper side of bended stems, the expression of PtFLAs was significantly increased in the TW xylem tissues compared to that in the OW xylem and the xylem tissues of normally grown plants [fig_ref] Figure 1: PtFLA6 expression analyses during TW formation [/fig_ref]. These results are highly consistent with previous reports about the expression pattern of the TW-associated FLA genes in poplar [bib_ref] Transcriptional and hormonal regulation of gravitropism of woody stems in Populus, Gerttula [/bib_ref] [bib_ref] Poplar genes encoding fasciclin-like arabinogalactan proteins are highly expressed in tension wood, Lafarguette [/bib_ref] [bib_ref] Biosynthesis of cellulose-enriched tension wood in Populus: global analysis of transcripts and..., Andersson-Gunnerås [/bib_ref] [bib_ref] Transcriptome profiling the basal region of poplar stems during the early gravitropic..., Azri [/bib_ref] [bib_ref] G-fibre cell wall development in willow stems during tension wood induction, Gritsch [/bib_ref]. To date, the tissue and cellular locations of poplar TW-associated FLAs have not been well reported. PtFLAs shared high identity in their amino acid sequences and similar size of protein molecular weight 21 , indicting that PtFLA6 polyclonal antibody may be able to identify all PtFLA6-like proteins, including other PtFLAs. Using PtFLA6 polyclonal antibody, we investigated the exact position of PtFLAs during TW by immunohistochemical analyses. PtFLA6 like proteins were abundantly accumulated in the DGF undergoing G-layer biosynthesis, and decreased as G-layer matured [fig_ref] Figure 2: Immunohistochemical analysis of PtFLA6 during TW formation [/fig_ref]. This result was supported by the findings of more accurate analyses using immunogold labeling TEM of PtFLA6 [fig_ref] Figure 3: Immunocytolocalization of PtFLA6 in the G-fiber cells of TW xylem tissues [/fig_ref]. The reduction of PtFLA proteins in mature GF is also consistent with a previous report showing that AGPs are strongly labeled in the secondary cell wall and less present in the G-layer of TW xylem fibers [bib_ref] Poplar genes encoding fasciclin-like arabinogalactan proteins are highly expressed in tension wood, Lafarguette [/bib_ref]. In addition, we also observed that the molecular weight of mature PtFLA6 detected in poplar stems was larger than that of its native protein. This could be caused by the glycosylation during the maturing of PtFLA protein. Indeed, five N-glycosylated sites and eleven O-glycosylated sites were predicted in PtFLA6 using the network prediction software [bib_ref] Antisense expression of the fasciclin-like arabinogalactan protein PtFLA6 gene in Populus inhibits..., Wang [/bib_ref].
Although the TW-associated FLAs in trees have been identified for decades, their biological functions are still unknown. Using the bended PtFLA6 antisense transgenic poplar grown in greenhouse, we found that PtFLAs play important roles in TW formation and differentiation. Reducing the expressions of PtFLAs and PtFLA6 proteins in the xylem tissues of the TW side resulted in a repressed TW formation [fig_ref] Figure 4: TW formation was inhibited in PtFLA6 antisense transgenic plants [/fig_ref] and more AGFs with thinner G-layer [fig_ref] Figure 4: TW formation was inhibited in PtFLA6 antisense transgenic plants [/fig_ref]. These results are consistent with the TW-specific expression pattern of PtFLA genes and the location of PtFLA6 like proteins in DGF during TW formation. However, PtFLAs did not affect the G-layer thickness [fig_ref] Figure 2: Immunohistochemical analysis of PtFLA6 during TW formation [/fig_ref]. Although this has fallen out of our expectation, it is easy to understand based on the protein location test showing that PtFLA6 proteins were less presented in the G-layer of MGF [fig_ref] Figure 3: Immunocytolocalization of PtFLA6 in the G-fiber cells of TW xylem tissues [/fig_ref]. It is note worthy that even the expressions of most PtFLAs were reduced by 40% to 80% in the TW tissues of PtFLA6 antisense transgenic lines [fig_ref] Figure 4: TW formation was inhibited in PtFLA6 antisense transgenic plants [/fig_ref] , TW formation was not severely or completely inhibited. This could be due to the redundant functions of PtFLAs, whose CDS are highly conserved [bib_ref] Antisense expression of the fasciclin-like arabinogalactan protein PtFLA6 gene in Populus inhibits..., Wang [/bib_ref] , and the residual transcripts of PtFLAs (PtFLA1/2/3/5/6/7/9/10), which are abundant enough for TW formation in transgenic plants.
It is widely considered that the G-layer, in the inner side of TW xylem, is a central part to generate the tensile force to push the inclined stem back to the vertical position. Using three-point tests, we also found that the flexural strength and stiffness of the bended stems with TW formation were all decreased in transgenic plants compared to that in WT plants [fig_ref] Figure 5: Asymmetrical distribution of GA 3 during TW formation [/fig_ref]. FLAs contain one or more cell adhesion domains (fasciclin domain), which work as adhesion molecules between the macromolecules of secondary cell wall by attaching to the plasma membrane [bib_ref] The fasciclin-like arabinogalactan proteins of Arabidopsis. A multigene family of putative cell..., Johnson [/bib_ref]. We speculate that PtFLAs located in the DGF may be responsible for the tensile force in the outer side of TW xylem.
As an important class of plant hormones, GA participates in the differentiation and elongation of xylem fibers and lateral root formation in trees [bib_ref] Interaction between indole-acetic acid and gibberellic in cambial activity, Wareing [/bib_ref] [bib_ref] The effect of applied growth hormones on cambial division and the differentiation..., Digby [/bib_ref] [bib_ref] Fiber length and gibberellins A1 and A20 are decreased in Eucalyptus globulus..., Ridoutt [/bib_ref] [bib_ref] Increased gibberellin biosynthesis in transgenic trees promotes growth, biomass production and xylem..., Eriksson [/bib_ref] [bib_ref] Gibberellins regulate lateral root formation in Populus through interactions with auxin and..., Gou [/bib_ref]. Previous studies have found that GA could play a positive role during TW formation, based on the observations that TW formation was induced or inhibited by the treatments using exogenous GA or GA biosynthesis inhibitors 11, 32-34 . We found that GA 3 and PttGA20ox1 transcripts were asymmetrically distributed in the upper side of bended stems undergoing TW formation [fig_ref] Figure 4: TW formation was inhibited in PtFLA6 antisense transgenic plants [/fig_ref]. These results are similar to the previous report that strong expression of PttACO1 (an ACC oxidase gene) and ethylene biosynthesis activity in TW xylem were detected 9 . Furthermore, daminozide treatment could inhibit GA 3 biosynthesis by repressing the expression of PttGA20ox1 in the TW tissues, resulting in a severe inhibited TW formation [fig_ref] Figure 6: TW formation was inhibited by daminozide treatment [/fig_ref]. Correspondingly, TW formation was also obviously suppressed in poplar plants treated with an ethylene perception inhibitor (1-methylcyclopropene, 1-MCP) [bib_ref] Ethylene is an endogenous stimulator of cell division in the cambial meristem..., Love [/bib_ref]. Therefore, GA may be another important plant hormone involved in regulating poplar TW formation. In addition, the transcription levels of PtFLAs were induced by exogenous GA 3 treatment [fig_ref] Figure 5: Asymmetrical distribution of GA 3 during TW formation [/fig_ref] , which is coincident with a recent report showing that GA could promote the expressions of poplar FLAs 7 . Our data revealed that the high level of GA 3 in the TW tissues of the bended stems or the decreased level of GA 3 in the inhibited TW by daminozide treatment was accompanied with up-or down-regulated PtFLAs, suggesting that GA 3 is an upstream regulator of PtFLAs.
The next question is how GA 3 regulates PtFLAs? As we know that GA signaling is mediated by the conserved repressors: DELLA proteins [bib_ref] Molecular mechanism of gibberellin signaling in plants, Sun [/bib_ref] [bib_ref] The molecular mechanism and evolution of the GA-GID1-DELLA signaling module in plants, Sun [/bib_ref]. GAs bind to the GA receptor GIBBERELLIN INSENSITIVE DWARF1 (GID1) to form a GA-GID1 complex, which immediately triggers the rapid degradation of DELLA proteins, then GA-induced genes are activated to complete the GA-regulated plant development and growth [bib_ref] Gibberellin-GID1-DELLA: a pivotal regulatory module for plant growth and development, Sun [/bib_ref]. The relationship between the repressed TW formation by daminozide, which severely reduced GA 3 content, and the inhibited degradation of PtRGA1 suggests that GA 3 -regulated TW formation is meditated by the DELLA protein PtRGA1 [fig_ref] Figure 6: TW formation was inhibited by daminozide treatment [/fig_ref]. DELLAs act as repressors in GA-mediated responses by interacting with other transcription factors and blocking their DNA binding or transactivation activities [bib_ref] A pivotal role of DELLAs in regulating multiple hormone signals, Davière [/bib_ref]. However, DELLA-DNA interaction may be indirect or less efficient by analyzing the RGA binding region of the promoter of its target genes using ChIP-qPCR test [bib_ref] Global analysis of Della direct targets in early gibberellin signaling in Arabidopsis, Zentella [/bib_ref]. Therefore, we performed tobacco transient expression assays and overexpressed PtRGA1 in xylem protoplasts to demonstrate the negative regulation of PtFLAs by PtRGA1.
Taken together, this study illustrates the biological function of PtFLAs in TW formation, as well as the regulatory mechanism of GA to PtFLAs. Although, more evidence is needed to completely understand the mechanism of GA and its signaling in TW formation, our data presented here imply a possible model of TW formation: under normal growth condition, the poplar DELLA protein PtRGA1 suppresses the transcriptions of PtFLAs, and then inhibits the TW formation [fig_ref] Figure 8: A proposed model of GA regulated TW formation [/fig_ref] ; when poplar plants are subjected to mechanical or gravity stresses, more GA 3 is produced in the xylem of upper side of bended or tilted stems to trigger the degradation of PtRGA1, and as a result, expressions of PtFLAs are activated to facilitate TW formation [fig_ref] Figure 8: A proposed model of GA regulated TW formation [/fig_ref].
# Methods
Plant materials and growth conditions. Populus trichocarpa genotype Nisqually-1 was used for the cloning of genes and promoters. Wild type Shanxin yang (P. davidiana × P. bolleana), a commercial hybrid clone, was used in every experiment as control in this study. The PtFLA6 antisense transgenic plants were generated by transforming the antisense PtFLA6 vector into Shanxin yang as described in our previous report [bib_ref] Antisense expression of the fasciclin-like arabinogalactan protein PtFLA6 gene in Populus inhibits..., Wang [/bib_ref] TW induction and histochemical staining. Before the induction of TW, WT and transgenic poplar plants grown in greenhouse were fixed to the sticks to make sure all the plants grow straight. The two-month-old poplar plants were bended at the twelfth internode (count from the top) to a 45° angle and fixed with cotton ropes to induce TW formation. After treated for 2 weeks, TW was well produced in the upper side of the bended stems and detected by histochemical staining as described below.
About 0.5 cm thick segments of bended internodes were fixed overnight at 4 °C in FAA solution. The segments were subsequently, passed over a gradient ethanol series, and then embedded in paraffin. Ten micrometer thick sections were cut out with a rotary microtome, and stained with 1% aqueous safranin-O (MP, USA) and subsequently with 1% aqueous astra-blue (Santa Cruz, USA) as described previously [bib_ref] A simple method that uses differential staining and light microscopy to assess..., Srebotnik [/bib_ref]. TW was stained into blue color in the xylem tissues of the upper side of the bended stems. OW remained in red color in the lower side of the bended stems. Images were taken under the SMZ800 microscope (Nikon, Japan). TW area and the whole xylem area of the bended stems were measured using the microscope software NIS-Element D 3.1 (Nikon, Japan). Then, the TW to xylem area ratio was calculated to illustrate TW formation.
Antibodies. PtFLA6 polyclonal antibody was produced by ABclonal (http://www.immunogen.com.cn) as described previously [bib_ref] Antisense expression of the fasciclin-like arabinogalactan protein PtFLA6 gene in Populus inhibits..., Wang [/bib_ref]. Arabidopsis RGA1 polyclonal antibody was purchased from Agrisera (http://www.agrisera.com/). Plant actin monoclonal antibody and the secondary antibodies used in this paper were bought from ABclonal.
Immunohistochemical localization. Segments (0.5 cm) of the bended stems for two weeks were fixed overnight in 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS, pH 7.5). Sections (10 µm) were cut out and blocked with 5% bovine serum albumin (BSA) for 1 h at room temperature. Then the sections were incubated with the PtFLA6 antibody (diluted at 1:50 with 0.1 M PBS containing 0.1% BSA) at room temperature for 1 h. After rinsed three times in PBS (5 min each), the samples were immersed in the alkaline phosphatase-conjugate secondary antibody (diluted at 1:100) for 1 h and then stained with 150-200 µl of Western Blue (Promega, USA). When blue color appeared, images were captured under bright field using the ECLIPSE 80i microscope (Nikon, Japan) or SMZ800 microscope (Nikon, Japan). Pre-immune serum was used as the negative control.
Quantitative real-time RT-PCR. For expression analysis of PttGA20ox1 during TW formation, the bended stems were cut into two parts from the division of TW side and OW side as shown in [fig_ref] Figure 1: PtFLA6 expression analyses during TW formation [/fig_ref]. Then the bark tissues stripped from the TW part and OW part (TW-B or OW-B) as well as the remnant xylem tissues with a part of pith (TW-X and OW-X) were collected to extract total RNA with the RNAiso Reagent (Takara, Japan). To detect the expression level of PtFLAs in transgenic plants during TW formation, the xylem tissues separated from the TW side were for RNA extraction. A total amount of 2 μg RNA was subjected to reverse transcription reaction using the HiScript ® II Q RT SuperMix for qPCR (+gDNA wiper) (Vazyme, China). qRT-PCR was performed using a AceQ qPCR SYBR Green Master Mix (Vazyme, China) and a CFX Connect Real-Time System (Bio-Rad, USA). The elongation factor gene PtEF1β was employed as an internal control in this study. And the relative expression of each target gene was normalized using PtEF1β. Gene specific primers used in this study were listed in . PttGA20ox1 primers were used as described previously PtFLA6 promoter activity analysis. To construct the promoter-GUS vector, the PtFLA6 promoter (~1.5 kb) was cloned from Nisqually-1 and inserted into in pBI121 by replacing the 35S promoter. The resultant construct was introduced into Shanxin yang by Agrobacterium-mediated transformation as described previously [bib_ref] An efficient Agrobacterium-mediated transformation and regeneration system for leaf explants of two..., Wang [/bib_ref]. The segments of bended stems of PtFLA6 promoter-GUS transgenic plants were used for GUS staining as described previously [bib_ref] An efficient Agrobacterium-mediated transformation and regeneration system for leaf explants of two..., Wang [/bib_ref]. Then the segments were fixed in FAA solution and embedded in paraffin. Sections (10 µm) were cut out and observed under bright field with the SMZ800 microscope (Nikon, Japan).
Western blotting. Total proteins were extracted using RIPA buffer, separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then electrotransfered onto polyvinylidene difluoride membranes. After blocked in TBST buffer supplemented with 5% non-fat dried milk for 1 hour at room temperature, the membranes were incubated with anti-PtFLA6 antibodies or anti-RGA1 (diluted at 1:1000) for 1 hour at room temperature. Afterwards, the membranes were rinsed three times with TBST buffer and subsequently incubated with the secondary antibodies (at a dilution of 1:5000) for 1 hour at room temperature. Then the membranes were put in the chemiluminescence detection solution LumiGLO (KPL, USA) and detected using the chemiluminescence image system Tanon 5500 (Tanon, China).
For western blotting analyses of PtFLA6 and poplar RGA1 during TW formation, xylem (X) and bark (B) tissues in the TW and OW sides of WT were separated for protein extraction. To detect PtFLA6 accumulation in transgenic TW, TW xylem tissues were used for protein extraction.
## Tem and immunogold labeling tem. for tem (transmission electron microscopic) observation, tw
tissues from the bended stems were fixed in glutaraldehyde solution, and embedded in EPON 812 resin (Shell, Holland). Sections (80 to 70 nm) were cut out, post-stained with uranyl acetate and lead citrate, and then observed with an H-7650 electron microscope (HITACHI, Japan).
For immunogold labeling TEM analyses, sections on grids were incubated with anti-PtFLA6 antibodies (diluted at 1:50). After hybridized with goat anti-rabbit gold conjugate (10 nm; Sigma, USA), the grids were washed and stained with 2% aqueous uranyl acetate. Control sections were obtained by omitting the primary antibody. GA 3 content assays. GA 3 extraction was performed as described previously with minor modification 31 .
Initially, freeze-dried materials were extracted with 80% (v/v) methanol containing the internal standard of [ 2 H 2 ]-GA 3 . The aqueous phase was extracted and then purified on C [bib_ref] Poplar genes encoding fasciclin-like arabinogalactan proteins are highly expressed in tension wood, Lafarguette [/bib_ref] Sep-Pak and MCX SPE columns (Qasis; Waters, USA). The eluant was dried, re-dissolved in HPLC initial solution, and then detected with a liquid chromatography-mass spectrometry system (Thermo, USA). Tandem mass spectrometry data were analyzed using the Xcalibur 2.1 software (Thermo, USA).
Exogenous GA 3 and daminozide treatments. Stem sections about 1 mm thick were sliced from the upper part stems of two-month-old wild type poplar grown in greenhouse. Then, the sections were soaked in 50 and 100 μM GA 3 aqueous solution, respectively. After 4 hours, the stem sections were used for qRT-PCR analysis of PtFLA expressions. Sections treated with water were used as control.
For GA biosynthesis inhibitor treatments, lanolin containing 5 mg ml −1 daminozide (Sigma, USA) was painted on the upper face of the bended stems of wild type plants. After two weeks, the treated stems were used for histochemical staining. Meanwhile, the TW side tissues were cut out and used for qRT-PCR, GA 3 content determination, and Western blotting. The bended stems pained with lanolin alone were used as control.
Tobacco transient expression assay. The promoters (~1.5 kb) of PtFLA1, PtFLA6 and PtFLA9 were amplified from the genomic DNA of Nisqually-1 and inserted into pGreenII0800-LUC 40 , respectively. The poplar DELLA protein gene PtRGA1, the homology of AtRGA1, was cloned from cDNA of Nisqually-1 to construct the pCAMBIA-2301 overexpression vector, driven by two copies of cauliflower mosaic virus 35S promoter [bib_ref] Study on expression character of gas gene contained in two different plasmids..., Huang [/bib_ref]. The constructs were introduced into Agrobacterium tumefaciens GV3101 (pMP90) by the freeze-thaw method.
Tobacco transient expression assays were performed as previously described [bib_ref] An intrinsic microRNA timer regulates progressive decline in shoot regenerative capacity in..., Zhang [/bib_ref]. Agrobacterium was re-suspended in infiltration buffer (10 mM methylester sulfonate, 10 mM MgCl 2 , and 150 μM acetosyringone, pH 5.7) at OD600 = 0.8 and then infiltrated into the tobacco leaves. After 3 days, Luciferin (1 mM) was painted on the transfected leaves before LUC activity was monitored. LUC signal was photographed with a chemiluminescence image system Tanon 5500 (Tanon, China). Three independent experiments (biological triplicates) were performed.
Transient expression of PtRGA1 in poplar xylem protoplasts. PtRGA1 CDS was inserted into pGreenII62-SK driven by the CaMV 35S promoter [bib_ref] Transient expression vectors for functional genomics, quantification of promoter activity and RNA..., Hellens [/bib_ref]. Then, stems of two-month-old poplar plants normally grown in greenhouse were used to isolate protoplasts. Xylem protoplasts isolation and cells transfection were performed as described previously [bib_ref] A simple improved-throughput xylem protoplast system for studying wood formation, Lin [/bib_ref]. Transfected protoplasts were used to for RNA extraction and qRT-PCR analyses of PtFLAs as described above.
[fig] Figure 1: PtFLA6 expression analyses during TW formation. (a) TW induction by mechanical stress. Twomonth-old wild type poplar plants (Shanxin yang) grown in greenhouse were bended for two weeks. The black vertical bar indicates the part to be taken for analyses. Scale bar = 1 cm. (b) Sections of bended stems were stained with safranin-O and astra-blue. TW was stained into blue. The horizontal line indicates the division between TW side and OW side. TW, tension wood; OW, opposite wood. Scale bar = 1 mm. (c) Western blotting analysis of PtFLA6 protein during TW formation. A total amount of 15 µg proteins was separated by SDS-PAGE and hybridized with anti-PtFLA6 antibodies (Anti-FLA6) or plant actin antibodies (Anti-Actin). FLA6 + GST, fusion protein for antibody production; OW-X, xylem tissues in the OW side; OW-B, the bark tissues of OW side; TW-X, xylem tissues in the TW side; TW-B, the bark tissues of TW side. (d) PtFLA6 promoter activity analysis during TW formation. Sections from the bended site of PtFLA6 promoter-GUS transgenic plants were used for GUS staining. Scale bar = 1 mm. [/fig]
[fig] Figure 2: Immunohistochemical analysis of PtFLA6 during TW formation. (a,b) Cross-sections of the bended stems of the wild type poplar plants were hybridized with anti-PtFLA6 antibodies (a) or pre-immune IgG as control (b). Green-brown signals of PtFLA6 were specifically detected in the TW. The horizontal line indicates the division between TW side and OW side. TW, tension wood; OW, opposite wood. Scale bar = 1 mm. (c) A higher magnification of the framed area in (a). REZ, radial expansion zone; DGF, differentiating G-fiber; MGF, mature G-fiber. (d-g) Higher magnification images of the framed areas in (c). PtFLA6 accumulation was decreased as the G-fiber maturing. Ve, vessel cell; Xf, xylem fiber cell. Scale bars = 25 μm. [/fig]
[fig] Figure 3: Immunocytolocalization of PtFLA6 in the G-fiber cells of TW xylem tissues. Transverse ultra thin sections of the xylem in the TW side were used for immunogold labeling TEM analysis. (a,d,g) Accumulation of PtFLA6 protein was examined in the differentiating G-fibers (a), the mature G-fibers (d) and the negative control (g), respectively. (b,e,h) Higher magnification images of the framed areas in (a,d,g), respectively. PtFLA6 labeling (black dots) was mainly detected in the differentiating G-fibers compared to that in the mature G-fibers. No labeling was seen in the control. (c,f,i) Higher magnification images of the framed areas in (b,e,h). PW, primary wall; SW, secondary wall; G, G-layer. Scale bars = 250 nm.SCIENtIfIC RePoRTS | 7: 6182 | DOI:10.1038/s41598-017-06473-9 [/fig]
[fig] Figure 4: TW formation was inhibited in PtFLA6 antisense transgenic plants. (a) Expression analysis of PtFLAs in the transgenic TW xylem. The expression of PtFLAs in the TW xylem of WT was set to 1. Error bars represent SDs from three biological replicates. (b) Western blotting analysis of PtFLA6 protein in the transgenic TW xylem. An amount of 30 μg proteins were separated by 10% SDS-PAGE and hybridized with PtFLA6 antibodies (Anti-FLA6) or plant actin antibodies (Anti-Actin), respectively. WT, wild type; L4 and L14, transgenic lines 4 and 14. (c) Ratio of TW area in the xylem area of transgenic plants. Error bars represent the SDs from five plants. **Indicates significant difference in comparison to WT at P < 0.01 (Student's t-test). (d) Histochemical staining analysis of TW formation in transgenic plants. The horizontal line indicates the division between TW side and OW side. TW, tension wood; OW, opposite wood. Scale bars = 1 mm. (e) Transmission electron micrographs of G-fibers in transgenic TW. More AGF were present in transgenic TW. GF, G-fiber; AGF, abnormal G-fiber with stunted G-layer production; G, G-layer. Scale bars = 10 μm. SCIENtIfIC RePoRTS | 7: 6182 | DOI:10.1038/s41598-017-06473-9 [/fig]
[fig] Figure 5: Asymmetrical distribution of GA 3 during TW formation. (a) Expression analysis of PtFLAs in GA 3 treated poplar stem sections. The expression level of genes in control was set to 1. (b) qRT-PCR analysis of PttGA20ox1 during TW formation. Gene expression in the OW xylem was set to 1. (c) GA 3 content in the xylem tissues of the TW and OW sides. ***Indicates a significant difference in comparison to OW-X at P < 0.001 (Student's t-test). (d) Western blotting analysis of the poplar DELLA protein in the xylem of TW and OW sides. About 30 μg proteins were separated by 10% SDS-PAGE and hybridized with AtRGA1 antibodies (Anti-RGA1) or plant actin antibodies (Anti-Actin), respectively. TW-B, the bark tissues of TW side; TW-X, xylem tissues in the TW side; OW-B, the bark tissues of OW side; OW-X, xylem tissues in the OW side. Error bars represent the SDs from three biological replicates. [/fig]
[fig] Figure 6: TW formation was inhibited by daminozide treatment. (a) Cross-sections of bended stems treated with daminozide or lanolin alone. The horizontal line indicates the division between TW side and OW side. TW, tension wood; OW, opposite wood. Scale bars = 1 mm. (b) Radio of TW area in the xylem of bended stems treated with daminozide. Error bars represent the SDs from five plants. (c) Expression analysis of PttGA20ox1 in the TW side of bended stems treated with daminozide. (d) GA 3 content in the TW side of daminozide treated stems. (e) Western blotting analysis of the poplar DELLA protein and PtFLA6 protein in the xylem of the TW side. An amount of 30 μg plant proteins extracted from the TW xylem tissues of bended stems treated with daminozide or lanolin were separated by 10% SDS-PAGE and hybridized with AtRGA1 antibodies (Anti-RGA1), PtFLA6 antibodies (Anti-FLA6) or plant actin antibodies (Anti-Actin). (f) qRT-PCR analysis of PtFLAs in the TW xylem tissues of bended stems treated with daminozide. Gene expression level in control was set to 1. Error bars represent the SDs from three biological replicates. Control, bended stems painted with lanolin alone; D, bended stems painted with daminozide. **Indicates a significant difference in comparison to control at P < 0.01 (Student's t-test). [/fig]
[fig] Figure 7: PtRGA1 negatively regulates the expression of PtFLAs. (a) Diagrams of effector and reporter constructs for transactivation studies. (b) Transactivation assays. Promoter activities of PtFLA1, PtFLA6 and PtFLA9 were obviously repressed in N. benthamiana leaves infiltrated with the construct combinations of Pro35S::PtRGA1 and each ProPtFLA::LUC. Bars = 1 cm. (c,d) Expression analysis of PtFLAs in the poplar xylem protoplasts overexpressing PtRGA1. RNA was extracted from the poplar xylem protoplasts transfected with pGreenII62-SK-PtRGA1. Then qRT-PCR was performed to analyze the transcriptional levels of PtRGA1 (c) and PtFLAs (d). Gene expression level in control was set to 1. Error bars represent the SDs from three biological replicates. Control, protoplasts transfected with pGreenII62-SK; PtRGA1, protoplasts transfacted with pGreenII62-SK-PtRGA1. SCIENtIfIC RePoRTS | 7: 6182 | DOI:10.1038/s41598-017-06473-9 [/fig]
[fig] Figure 8: A proposed model of GA regulated TW formation. (a) The poplar DELLA protein PtRGA1 inhibits the expression of PtFLAs under normal growth, and then represses the TW formation. (b) When submitted to mechanical or gravity stress, more GA 3 will be produced on the upper side of the bended or tilted poplar stems, and then triggers the degradation of PtRGA1 proteins, which promotes PtFLAs transcription to facilitate TW formation. [/fig]
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Is nab-paclitaxel better than conventional taxanes as neoadjuvant therapy for breast cancer? A meta-analysis
Objective: This study compared the efficacy and safety of nanoparticle albumin-bound paclitaxel (nab-paclitaxel) with conventional taxanes as neoadjuvant chemotherapy for breast cancer. Methods: We searched the literature using PubMed, the Cochrane Library, and Web of Science from their inception to December 15, 2019 based on predetermined inclusion and exclusion criteria. The relevant studies compared pathologic complete response (pCR) and adverse event rates. Results: The meta-analysis included five studies and 2335 patients. Compared with conventional taxanes, neoadjuvant chemotherapy with nab-paclitaxel was associated with a higher pCR rate (odds ratio [OR] ¼ 1.39, 95% confidence interval [CI] ¼ 1.16-1.67), especially among patients with triple-negative breast cancer or Ki67 indices of >20%. Pooled outcomes also revealed better event-free survival in the nab-paclitaxel group (hazard ratio ¼ 0.69, 95% CI ¼ 0.57-0.85). However, all-grade (OR ¼ 2.17, 95% CI ¼ 1.38-3.40) and grade !3 peripheral sensory neuropathy (OR ¼ 3.92, 95% CI ¼ 2.44-6.28) were more frequent in the nab-paclitaxel group.Conclusions: This meta-analysis implied that nab-paclitaxel more effectively improved pCR than conventional taxanes. Nab-paclitaxel may have greater benefits in patients with triple-negative breast cancer. However, additional attention is required for the early diagnosis and management of peripheral sensory neuropathy.
# Introduction
Neoadjuvant chemotherapy (NAC) is an important treatment option for invasive breast cancer. Some studies observed similar efficacy between adjuvant and neoadjuvant therapy.Taxanes and anthracyclines are two of the most of effective chemotherapeutic classes used as NAC in breast cancer. The conventional taxanes include docetaxel and paclitaxel. The NSABP B27 study found that the addition of docetaxel notably increased the pathologic complete response (pCR) rate from 13.7 to 26.1%.Nanoparticle albumin-bound paclitaxel (nab-paclitaxel) is a novel taxane that was first approved by the FDA for its superior efficacy compared with solvent-based paclitaxel in the treatment of metastatic breast cancer. Nab-paclitaxel allows the use of larger drug doses without premedication, e.g., 260 mg/m 2 versus 175 mg/m 2 for paclitaxel.However, another phase III trial found that nab-paclitaxel was not superior with a trend toward inferiority, and toxicity was increased in the metastatic setting.There is no clear consensus concerning whether nab-paclitaxel is more effective than conventional taxanes in the treatment of metastatic breast cancer. The same is also true for the application of nabpaclitaxel as NAC in breast cancer. Two randomized clinical trials reported different results.Early single-arm studies on the efficacy of nab-paclitaxel in NAC for breast cancer were summarized in a metaanalysis.However, because of the small number of comparative studies, the metaanalysis extracted data from only three studies and pooled the results from two studies to evaluate the safety of these two generations of taxanes. In addition, the study did not explore which individuals can benefit from nab-paclitaxel.
More recently, the results of some comparative studies have been updated. Therefore, we conducted this meta-analysis to compare efficacy and safety between nabpaclitaxel and conventional taxanes.
# Materials and methods
We conducted this meta-analysis following the recommendations of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses. 11
## Literature search strategy
We performed a literature search using PubMed, the Cochrane Library, and Web of Science from inception to December 15, 2019 using predetermined inclusion and exclusion criteria without language limitations. We searched the terms (("albuminbound paclitaxel" or "nab-paclitaxel" or "ABI-007" or "Abraxane") and ("Taxanes" or "paclitaxel" or "docetaxel") and ("neoadjuvant" or "preoperative") and ("breast cancer")) using the Boolean 'AND' operator. Two reviewers (W. Li and Lu) independently analyzed all potentially relevant studies. In the case of any disagreements, consensus was reached by discussion. The same method was also used for quality assessment of the selected studies.
## Inclusion and exclusion criteria
The included studies compared nabpaclitaxel with conventional taxanes (paclitaxel or docetaxel) as NAC for patients with non-metastatic breast cancer. The two arms in the studies had the same chemotherapy regimens excluding the taxanes used. Studies had clear definitions of pCR. Editorials, case reports, letters to the editor, review articles, and studies with insufficient information for data extraction were excluded.
## Data extraction
Data from the included studies were extracted and summarized independently by two of the authors (Lu and Lin). Any disagreement was resolved by a third author (Y. Li).
The primary outcomes were the pCR and adverse event rates. If sufficient data were available, then the pCR rate for invasive subtypes of breast cancer was extracted, such as triple-negative breast cancer (TNBC; negative for estrogen receptor [ER], progesterone receptor [PR], and human epidermal growth factor receptor 2 [HER2]), HER2-positive breast cancer (HER2-positive regardless of the ER or PR status), and tumors with high Ki67 expression, defined as a Ki67 index exceeding 20%.
There are two common definitions of pCR in breast cancer, namely ypT0 ypN0 (no invasive or non-invasive residual tumor in the breast and axillary lymph nodes) and ypT0/is ypN0 (no invasive residual tumor in the breast and axillary lymph nodes). However, if we adopted either of the two definitions, some studies had to be abandoned, which would weaken the stability of the results because of the fewer numbers of studies and samples included.
Therefore, we extracted all pCR data using ypT0 ypN0 or ypT0/is ypN0 as the definition. This ensured the availability of more studies for each pooled analysis to choose pCR according to a definition employed in our included studies. Adverse events included hematologic toxicity, peripheral sensory neuropathy, fatigue, rash, nausea, allergic reactions, and elevated liver enzymes. Adverse events were categorized according to the National Cancer Institute Common Terminology Criteria for Adverse Events. Severe adverse events were defined as Grade 3 or higher. The secondary outcomes were the clinical overall response rate (ORR), overall survival (OS), and event-free survival (EFS). The events for EFS were disease progression during neoadjuvant therapy resulting in inoperability, any invasive locoregional recurrence of disease after neoadjuvant therapy, any invasive contralateral breast cancer, any distant recurrence of disease, or all-cause mortality, whichever occurred first.
## Quality assessment and statistical analysis
The quality of randomized controlled trials (RCTs) was assessed using the Cochrane risk of bias tool.The quality of retrospective studies was checked using the modified Newcastle-Ottawa Scale (NOS).RCTs and retrospective studies achieving eight or more stars on the NOS were considered to be of higher quality. Sensitivity analysis was performed for high-quality studies, and only outcomes reported in more than three studies were included in this analysis.
Because the definition of pCR differed among the studies, the adopted definition should include more studies or samples for data analysis. Odds ratios (ORs) were used to compare dichotomous variables. Hazard ratios (HRs) were used as summary statistics for OS and EFS. All results were reported with 95% confidence intervals (CIs). The random-effects model was used after exploring the causes of heterogeneity. Otherwise, the fixed-effects model was used.Two-tailed P < 0.05 with a 95% CI that did not include 1 was considered statistically significant. Statistical heterogeneity was calculated using Cochrane's Q statistic and the I 2 statistic, and significance was indicated by P < 0.1. Funnel plot asymmetry was assessed using Begg's and Egger's tests to evaluate publication bias, and two-tailed P < 0.05 was considered statistically significant.
Statistical analyses were performed using Review Manager 5.3 (Copenhagen: The Nordic Cochrane Centre, The Cochrane Collaboration) and Stata 12.0 (Stata Corporation, College Station, TX, USA).
# Results
Five studieswere identified using the predefined search strategy in the final analysis. Two 19,20 studies reported survival after short follow-up periods. The sample sizes ranged from 120 to 1206. In total, 2335 patients were eligible for the analysis, with 1140, 1118, and 77 patients receiving nabpaclitaxel, solvent-based paclitaxel, and docetaxel, respectively. The study characteristics, patient demographics, regimens, dosages, and quality score for each study are shown in. The analyzed studies included three RCTs,one retrospective study,and one matched casecontrol study.The details of the quality assessment are shown in.
## Primary outcomes
pCR. All studies reported pCR using the definition of ypT0/is ypN0 in the whole population, whereas ypT0 ypN0 was more frequently used in subtype analyses. Therefore, to obtain more stable results, we pooled the pCR data using the ypT0/is ypN0 definition in the global analysis and the ypT0 ypN0 definition in the subtypes analyses.
Pooling the data of all five studiesthat assessed pCR (ypT0/is ypN0) in 2335 patients, a significant difference favoring nab-paclitaxel was identified (OR ¼ 1.39, 95% CI ¼ 1.16-1.67; P < 0.001;with no heterogeneity found among the studies (v 2 ¼ 1.34,
[formula] I 2 ¼ 0%). [/formula]
Pooling the data from three studies 9,17,18 that assessed pCR (ypT0 ypN0) in 1029 patients with Ki67 > 20%, a significant difference favoring nab-paclitaxel was noted (OR ¼ 1.64, 95% CI ¼ 1.26-2.13, P < 0.001) with no significant heterogeneity observed among the studies (v 2 ¼ 0.58, I 2 ¼ 0). Pooling the data from three studies 9,17,18 that assessed pCR (ypT0 ypN0) among 364 patients with TNBC, a significant difference favoring nab-paclitaxel was observed (OR ¼ 2.45, 95% CI ¼ 1.57-3.82, P < 0.001) with moderate heterogeneity noted among the studies (v 2 ¼ 3.24, I 2 ¼ 38%). Pooling the data of two studies 9,18 that assessed pCR (ypT0 ypN0) in 449 patients with HER2-positive breast cancer, comparable efficacy was noted between the two groups (OR ¼ 1.31, 95% CI ¼ 0.89-1.92) with low heterogeneity found among the studies (v 2 ¼ 1.05, I 2 ¼ 5%).
Incidence of adverse events. We gathered adverse event data for neutropenia, peripheral sensory neuropathy, fatigue, rash, and vomiting at all grades. The frequency of allgrade vomiting was similar between the groups (OR ¼ were significantly more frequent in the nab-paclitaxel arm. However, there was significant between-study heterogeneity regarding the incidence of peripheral sensory neuropathy (v 2 ¼ 17.15,
[formula] I 2 ¼ 77%, P ¼ 0.002) [/formula]
. We found that the major source of heterogeneity originated from two studies that used a higher cumulative dose of nab-paclitaxel. Comparing nab-paclitaxel to conventional taxanes in terms of severe adverse events, there were no significant differences in the rates of neutropenia (OR ¼. There was a significant between-study heterogeneity concerning the incidence of severe neutropenia (v 2 ¼ 15.96, I 2 ¼ 81%, p ¼ 0.001); however, we failed to identify the source of heterogeneity.
## Secondary outcomes
ORR. Four studiesassessed the clinical ORR in 2173 patients. The clinical ORR was not significantly different between the nab-paclitaxel and conventional taxane groups (OR ¼ 1.19, 95% CI ¼ 0.97-1.46), and no significant heterogeneity was noted among the studies (v 2 ¼ 0.96, I 2 ¼ 0%;.
## Efs.
Among the included studies, two studies reported EFS data,and only one study reported OS.The meta-analysis illustrated that nab-paclitaxel was associated with significantly better EFS than conventional treatment (HR ¼ 0.69, 95% CI ¼ 0.57-0.85, P < 0.001), and moderate between-study heterogeneity was observed (v 2 ¼ 1.91, I 2 ¼ 48%).
## Sensitivity analysis and publication bias
Three RCTsand one retrospective studywith NOS scores of at least eight stars were included in the sensitivity analysis, which revealed no change in the significance of any of the outcomes.presents a funnel plot of the studies included in this meta-analysis that reported pCR. All studies lied inside the 95% CIs with an even distribution around the vertical, indicating no obvious publication bias. Neither Begg's funnel plot nor Egger's test indicated the existence of publication bias.
# Discussion
This meta-analysis comparing nabpaclitaxel and conventional taxanes as NAC for breast cancer revealed that nabpaclitaxel was linked to better pCR. Both severe and all-grade peripheral sensory neuropathy were more common in the nab-paclitaxel group. Because no change was found in the sensitivity analysis of high-quality studies, the stability of the results was confirmed.
Among the included studies, two large RCTs recently reported short-term followup results.The pooled EFS data favored nab-paclitaxel. A meta-analysis by CTNeoBC demonstrated that patients with pCR had better EFS.We therefore speculated that the better EFS in the nabpaclitaxel group could be attributable to the higher pCR rate. Nonetheless, we also noted that the clinical ORR was comparable between the two groups, in line with the results of another meta-analysis focusing on metastatic breast cancer.This study demonstrated that patients with TNBC or Ki67 > 20% could more greatly benefit from nab-paclitaxel. This finding has considerable clinical implications for the clinical treatment of TNBC given that chemotherapy is the main adjuvant treatment for TNBC. However, whether nab-paclitaxel has benefits for the treatment of other types of breast cancer remains obscure. Most breast cancers treated with NAC in clinical practice have Ki67 > 20%. In addition, several clinicopathological parameters are significantly associated with the Ki67 index, such as the molecular subtype, lymph node status, tumor size, tumor grade, and TNM stage.Concerning HER2-positive breast cancer, the result must be interpreted with caution because of the small number of studies of HER2-positive breast cancer. However, there will be few opportunities to draw valid conclusions regarding this issue in further research. In this era of precision medicine, targeted therapy will attract more attention than chemotherapy for HER2positive breast cancer. As indicated by the design of the TEAL study, an RCT of early-stage HER2-positive breast cancer, although nab-paclitaxel and solvent-based paclitaxel were examined in different study arms, the researchers focused on comparisons of efficacy between two dual anti-HER2 therapeutic strategies (T-DM1 and lapatinib vs. trastuzumab and pertuzumab).Peripheral sensory neuropathy, rash, neutropenia, and fatigue were more frequent in the nab-paclitaxel arm than in the conventional taxane arm, but regarding severe events, only the rate of severe peripheral sensory neuropathy differed between the groups. Taxanes are microtubulestabilizing agents, and the major toxicity of these agents is peripheral sensory neuropathy.The mechanism of taxaneinduced peripheral sensory neuropathy remains unclear, but some risk factors may explain the higher risk of taxaneinduced peripheral sensory neuropathy. In a randomized phase III study, the mean cumulative dose to the onset of grade !2 peripheral sensory neuropathy was 371 mg/m 2 for docetaxel, versus 715 mg/m 2 for paclitaxel.The severity of peripheral sensory neuropathy is also related to the dose. In the CALGB 9342 trial, grade !3 peripheral sensory neuropathy was observed in 33% of patients who received paclitaxel at a dose of 250 mg/m 2 , 19% of patients treated at a dose of 210 mg/m 2 , and 7% of patients treated at a dose of 175 mg/ m 2 .Nab-paclitaxel uses a nanoparticle drug delivery system, thereby permitting higher dose delivery and intensity.This may explain why nab-paclitaxel is associated with a higher incidence of peripheral sensory neuropathy than conventional taxanes. Currently, there is no agent for preventing peripheral sensory neuropathy. Thereby, early detection and timely dose reduction can alleviate symptoms in patients with peripheral sensory neuropathy. This is consistent with the finding from the GeparSepto trial that proper dose reduction reduced the incidence and severity of peripheral sensory neuropathy without compromising treatment efficacy.This study had some potential limitations. First, only five studies were included, and some pooled analyses were performed with two or three studies. Second, the follow-up period was short; therefore, the evidence supporting the survival benefits of nab-paclitaxel is inadequate. Third, the patient cohorts, treatment strategies (concurrent or sequential administration), drug dosages, and administration interval varied among the studies, which may have led to bias. Finally, no language restrictions were applied in the literature search, but all available studies were published in English. Thus, clinical data reported in other languages may have been missed.
# Conclusions
Although further studies are needed to identify patients who might benefit from nab-paclitaxel, our meta-analysis clearly indicated that nab-paclitaxel is more effective than conventional taxanes as NAC in terms of pCR in breast cancer. Moreover, early diagnosis and intervention are needed to control peripheral sensory neuropathy during the administration of nab-paclitaxel.
# Author contributions
W. Li and Y. Li conceived and designed the study. W. Li and Lu conducted the literature selection and quality assessment. Lu, Lin, and Y. Li performed the data acquisition. Lu, Y. Li performed the statistical analysis. Y. Li provided the manuscript editing. All authors read and approved the final manuscript.
## Declaration of conflicting interest
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Selection of Bis-Indolyl Pyridines and Triphenylamines as New Inhibitors of SARS-CoV-2 Cellular Entry by Modulating the Spike Protein/ACE2 Interfaces
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the infectious agent that has caused the current coronavirus disease (COVID) pandemic. Viral infection relies on the viral S (spike) protein/cellular receptor ACE2 interaction. Disrupting this interaction would lead to early blockage of viral replication. To identify chemical tools to further study these functional interfaces, 139,146 compounds from different chemical libraries were screened through an S/ACE2 in silico virtual molecular model. The best compounds were selected for further characterization using both cellular and biochemical approaches, reiterating SARS-CoV-2 entry and the S/ACE2 interaction. We report here two selected hits, bis-indolyl pyridine AB-00011778 and triphenylamine AB-00047476. Both of these compounds can block the infectivity of lentiviral vectors pseudotyped with the SARS-CoV-2 S protein as well as wild-type and circulating variant SARS-CoV-2 strains in various human cell lines, including pulmonary cells naturally susceptible to infection. AlphaLISA and biolayer interferometry confirmed a direct inhibitory effect of these drugs on the S/ACE2 association. A specific study of the AB-00011778 inhibitory properties showed that this drug inhibits viral replication with a 50% effective concentration (EC 50 ) between 0.1 and 0.5 mM depending on the cell lines. Molecular docking calculations of the interaction parameters of the molecules within the S/ACE2 complex from both wild-type and circulating variants of the virus showed that the molecules may target multiple sites within the S/ACE2 interface. Our work indicates that AB-00011778 constitutes a good tool for modulating this interface and a strong lead compound for further therapeutic purposes.
S evere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a positive-strand RNA virus that causes severe respiratory syndrome in humans. While several vaccines have been developed since the beginning of the pandemic, no efficient drugs have been identified as having a strong effect on viral replication for use in the clinic. Therefore, a better understanding of the viral replication processes and the search for new antiviral approaches remain of interest. Viral entry into cells requires an interaction between the spike (S) protein of the virus and angiotensin-converting enzyme 2 (ACE2) at the surface of the cell membrane. ACE2 has been reported as a receptor for SARS-CoV-2 in the lung, as previously shown for SARS-CoV, which was responsible for the first SARS outbreak in 2003 [bib_ref] Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus, Li [/bib_ref]. ACE2 is expressed in human airway epithelia, lung parenchyma, vascular endothelia, kidney cells, and small intestine cells but is only weakly expressed in the brain [bib_ref] Tissue distribution of ACE2 protein, the functional receptor for SARS coronavirus. A..., Hamming [/bib_ref]. Thus, converging data from the literature indicate that the S/ACE2 interaction remains the central entry pathway for SARS-CoV-2. More recently, the expression of the cellular protease TMPRSS2 has been shown to dictate the entry route used by the virus to infect cells [bib_ref] TMPRSS2 expression dictates the entry route used by SARS-CoV-2 to infect host..., Koch [/bib_ref]. These entry routes, as well as the role of the S/ACE2 interaction in these processes, remain to be fully characterized. However, as the first contact point between the virus and the cell, the S/ACE2 complex constitutes an attractive target for blocking very early events of viral infection. Furthermore, in addition to being potential therapeutic agents, drugs targeting this association may constitute useful tools for dissecting the further processes of the entry step.
In this work, we used in silico, in vitro, and in cellulo models that allow the monitoring of the S/ACE2 complex [bib_ref] In silico, in vitro and in cellulo models for monitoring SARS-CoV-2 spike/human..., Lapaillerie [/bib_ref] and select drugs that may target the S/ACE2 interface from 139,146 compounds. Using these systems, we selected approximatively 20 potential S/ACE2 inhibitors, and among them, we identified two novel chemical series capable of specifically blocking the SARS-CoV-2 entry step by impairing the ACE2/S interaction, leading to inhibition of viral SARS-CoV-2 replication in naturally susceptible cells. This work also provides molecular details on the inhibitory mechanism, paves the way for a potential new chemical strategy to interfere with the early stage of viral entry, and reports the first druggable protein/protein inhibitor (PPI) series able to disrupt the S/ACE2 interaction.
# Results
In silico virtual screening of drugs targeting the S/ACE2 interfaces. To select potential drugs targeting the interaction between the spike protein and its cellular receptor, ACE2, we used the 100-ns molecular dynamics (MD) simulation previously performed [bib_ref] In silico, in vitro and in cellulo models for monitoring SARS-CoV-2 spike/human..., Lapaillerie [/bib_ref] with the AMBER force field and TIP3P water box with the published S/ACE2 complex [bib_ref] Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor, Lan [/bib_ref]. The identified transient interfaces within the S/ACE2 complex were used after incorporating the dynamic nature of the interface to determine the pockets to be explored during the virtual screening campaigns. This simulation allowed us to identify druggable sites within the S/ACE2 complex, which are reported in [fig_ref] FIG 1: Selection of drugs targeting the S/ACE2 interaction by virtual screening studies [/fig_ref] and B. These druggable sites were then used for the virtual drug screening steps. To this purpose, the Mu.Ta.Lig. Virtual Chemotheca (approximatively 60,000 molecules; http://chemotheca.unicz.it), the ZINC FDA Approved and ZINC In-Trials Database (1,379 and 5,811 molecules for evaluation for drug repurposing evaluation; https://zinc.docking.org), the French National Chemical Library (Chimiothèque Nationale , 70,000 molecules; https://chembiofrance.cn.cnrs.fr/fr/ composante/chimiotheque), and the Inhibitors of Protein-Protein Interactions Database (1,956 molecules; https://ippidb.pasteur.fr) were used. Among the 139,146 selected compounds, 200 molecules were first selected and classified based on their Vina and PLP scores when tested either on the full S/ACE2 complex [fig_ref] FIG 1: Selection of drugs targeting the S/ACE2 interaction by virtual screening studies [/fig_ref] or the S protein alone [fig_ref] FIG 1: Selection of drugs targeting the S/ACE2 interaction by virtual screening studies [/fig_ref]. The best compounds from each library were then extracted, allowing us to select approximatively 20 hits for further analyses (see the list of selected compounds in [fig_ref] TABLE 1: IC 50 , CC 50 , and SI values for AB-00047476 and... [/fig_ref] and their structure in [fig_ref] FIG 2: Effect of AB-00047476 [/fig_ref] , both in the supplemental material). Based on their originality and availability, we focused our present work on the molecules selected from the CN.
In cellulo screening for SARS-CoV-2 S/ACE2-mediated entry. The 10 best molecules from the CN (see their structure in [fig_ref] FIG 2: Effect of AB-00047476 [/fig_ref] were selected and first tested on the lentiviral vector-based cellular approach previously set up in the laboratory (6) using lentiviral vectors pseudotyped with the SARS-CoV-2 spike protein (CoV-2 LV) and HEK293T-ACE2 cells containing 9 copies of the ACE2 gene (see reference 6 and Materials and Methods below for a full description of the procedures). As reported in [fig_ref] FIG 3: Molecular docking of AB-00011778 and AB-00047476 to the spike/ACE2 complex and to... [/fig_ref] and B, the CoV-2 LVs were shown to efficiently transduce only the 293T-ACE2 cells, in contrast to the original 293T cells, leading to the integration and expression of the enhanced green fluorescent protein (eGFP) gene carried by the LV. This confirmed that the transduction efficiency indicated by the percentage of eGFP-positive cells relied on the early S/ACE2 interaction. In contrast, typical vesicular stomatitis virus G protein (vesicular stomatitis virus [VSV]-g) pseudotyped LVs able to transduce both 293T and 293T-ACE2 cells served as a specificity control for the next selection of drugs specific for S/ACE2 entry. Competition experiments using the soluble ACE2 protein fully validated the S/ACE2 dependency of LV CoV-2 infectivity, while LV VSV-g was not affected by the protein [fig_ref] FIG 3: Molecular docking of AB-00011778 and AB-00047476 to the spike/ACE2 complex and to... [/fig_ref].
The selected drugs were tested for both VSV-g and SARS-CoV-2 LV infectivity to determine their possible effects on the S/ACE2-mediated entry pathway. The first screening, using a 10 mM concentration of each compound, showed a significant inhibitory effect for certain molecules on CoV-2 LVs without any effect on VSV-g LVs [fig_ref] FIG 4: Effects of AB-00011778 and AB-00047476 on the in vitro S-RBD/ACE2 interaction using... [/fig_ref]. Among the selected drugs, AB-00074850, AB-00054798, AB-00047476, and AB-00011778 were found to be the most efficient by inducing a 40% to 80% decrease in CoV-2 LV viral infectivity without any effect on VSV-g LV. Further accurate analysis of the inhibitory effects of the molecules using increasing concentrations showed that AB-00074860 and AB-00054798 exhibited only a modest inhibition of CoV-2 LV infectivity at concentrations below 10 mM while AB-00047476 and AB-00011778 conserved a significant inhibition capability without any effect on VSVg LV [fig_ref] FIG 2: Effect of AB-00047476 [/fig_ref]. We thus decided to focus on these two molecules, AB-00047476 and AB-00011778. The cytotoxicity of the two compounds was then determined in different cell lines, including HEK293T, A549, Calu3, and Caco2, using the MTT 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide assay. No cytotoxicity was shown within the 0 to 30 mM range, while some slight decrease in cell viability could be observed at higher concentrations [fig_ref] FIG 5: Effects of AB-00014778 and AB-00047476 on the S-RBD/ACE2 interaction in a cellular... [/fig_ref].
Taken together, our data strongly suggest that both AB-00047476 and AB-00011778 interfered with the early S/ACE2-mediated SARS-CoV-2 entry process. To confirm this, we next investigated using molecular docking and biochemical approaches whether this inhibitory effect could be due to a direct effect of the drugs on the physical interaction between S and ACE2.
Molecular docking analysis of the drug interactions with S and ACE2. To better understand the binding of AB-00011778 and AB-00047476 to the target protein, molecular docking studies were performed. [fig_ref] FIG 3: Molecular docking of AB-00011778 and AB-00047476 to the spike/ACE2 complex and to... [/fig_ref] displays the results from the predicted binding poses to the interface between the minimal receptor binding domain from SARS-CoV-2 spike protein (S-RBD) and ACE2 and to the S-RBD alone, highlighting the interacting amino acid residues.
The docking of AB-00011778 to the S-RDB/ACE2 interface [fig_ref] FIG 3: Molecular docking of AB-00011778 and AB-00047476 to the spike/ACE2 complex and to... [/fig_ref] resulted in a score of 92.05. S-RDB residues Arg403, Gln409, and Lys417 establish strong hydrogen bonds with this molecule. Arg403 interacts with one oxygen from one of the sulfonyl groups with a distance of 3.00 Å. Gln409 and Lys417 interact with the nitrogen from the central pyridine with distances of 3.78 Å and 3.55 Å, respectively. In addition, the ACE2 His34 residue interacts with the indole group via p -p stacking interactions. ACE2 residues Lys26, Leu29, and Asp38 and S-RDB residues Lys417 and Gln493 are also involved in hydrophobic interactions with AB-00011778. The interactions of AB-00011778 with the amino acid residue Asp38 of ACE2 and with S-RDB Lys417 and Gln493 are of particular relevance. In fact, previous molecular docking studies of the S-RDB/ACE2 interface (6) have shown that Asp38 from ACE2 establishes important hydrogen bonds with S-RDB Tyr449 and Gln498, that S-RDB Lys417 was shown to be involved in very stable hydrogen bonds and charge interactions with Asp30 from ACE2, and that S-RDB Gln493 formed important hydrogen bonds with ACE2 Glu35. The disruption of these interactions caused by AB-00011778 binding should lead to a blockade of the S/ACE2 interaction and could be one of the reasons why this molecule is a successful inhibitor of this system.
The docking of AB-00011778 to the S-RBD [fig_ref] FIG 3: Molecular docking of AB-00011778 and AB-00047476 to the spike/ACE2 complex and to... [/fig_ref] in the absence of ACE2 led to a score of 89.12. The obtained docking pose shows the formation of a strong hydrogen bond between Gly502 and one of the AB-0001778 ether groups, with a length of 1.90 Å. There is also the formation of p -p stacking interactions between Tyr505 of the indole group and two of the ligand's phenyl groups. Finally, Lys417, Leu455, Asn501, and Tyr505 are involved in hydrophobic interactions with AB-00011778. Gly502 and Tyr505 have been implicated from molecular docking studies of the S-RDB/ACE2 interface to participate in very stable hydrogen bonds with ACE2, with Gly502 interacting with Lys353 and Tyr505 interacting with ACE2 Glu37, during 57% and 56% of the simulation times, respectively [bib_ref] In silico, in vitro and in cellulo models for monitoring SARS-CoV-2 spike/human..., Lapaillerie [/bib_ref]. Hence, the interactions of AB-00011778 with these two residues are likely to significantly interfere with the S-RDB/ACE2 association.
The docking of AB-00047476 to the S-RDB/ACE2 interface [fig_ref] FIG 3: Molecular docking of AB-00011778 and AB-00047476 to the spike/ACE2 complex and to... [/fig_ref] resulted in a score of 100.71 (GOLD/PLP). The most favored pose suggests that this molecule forms one hydrogen bond through the nitrogen of one of its three benzothiazole groups and S-RDB Arg403, with a distance of 2.14 Å. In addition, AB-00047476 forms hydrophobic interactions with ACE2 Asn33, Thr92, and Pro389 and with S-RDB Lys417 and Tyr495.
The docking of AB-00047476 to the S-RDB protein alone [fig_ref] FIG 3: Molecular docking of AB-00011778 and AB-00047476 to the spike/ACE2 complex and to... [/fig_ref] resulted in a score of 89.58. The most favored pose of this molecule reveals two hydrogen bonds: one between Arg403 and the nitrogen from one of the benzothiazole groups and another between Gly502 and the nitrogen from a second benzothiazole group, with lengths of 2.28 Å and 2.26 Å, respectively. There is also the formation of p -p stacking interactions between Tyr505 and one of the phenyl groups of the triphenylamine core in the center of the molecule. Residues Arg403, Lys417, Tyr449, and Tyr505 from the S-RDB are also involved in hydrophobic interactions with the molecule. The interactions of AB-00047476 with Tyr449, Gly502, and Tyr505 from the S-RDB are of particular relevance, as these residues have been shown to be important for the formation of the S-RDB/ ACE2 complex. A previous molecular docking simulation [bib_ref] In silico, in vitro and in cellulo models for monitoring SARS-CoV-2 spike/human..., Lapaillerie [/bib_ref] of the S-RDB/ACE2 complex showed that these residues of the spike protein are involved in important hydrogen bonds with ACE2. Tyr449 interacts with ACE2 Asp38, Gly502 interacts with ACE2 Lys353, and Tyr505 interacts with ACE2 Glu37. All of these are major interactions, being present during 54%, 57%, and 56% of the simulation times, respectively. Therefore, the results suggest that AB-00047476 binding to S-RDB would prevent the normal S-RDB/ACE2 association.
Additionally, the abilities of AB-00011778 and AB-00047476 to competitively bind to ACE2 with the spike protein was evaluated. Both molecules were docked into the ACE2 receptor, defining a potential binding region on the surface of ACE2 that is known to interact and recognize the spike protein. The results are presented in [fig_ref] FIG 6: Effects of AB-00011778 and AB-00047476 on SARS-CoV-2 replication in human pulmonary cells [/fig_ref] , showing that AB-00047476 and AB-00011778 can bind ACE2 with high binding scores that are comparable to those observed when these molecules were docked to the S-RDB/ACE2 interface and to S-RDB alone.
The docking of AB-00011778 to ACE2 alone resulted in a score of 81.41 (GOLD/PLP). The most stable pose reveals the formation of multiple hydrogen bonds between ACE2 and AB-00011778: (i) between Tyr41 and the nitrogen from the central pyridine group, with a length of 1.66 Å; (ii) between Gln325 and Gly326 interacting with the oxygen from one of the ligand's ether groups, with lengths of 2.04 Å and 3.09 Å, respectively; and (iii) between Asn330 and one oxygen from one of the sulfonyl groups, with a distance of 2.10 Å. One can also observe the formation of one p -cation interaction between Lys353 and the phenyl ring from one of the indole groups. Glu37, Tyr41, Thr324, Gln325, and Lys353 are involved in hydrophobic interactions. Glu37, Tyr41, and Lys335 have also been previously implicated [bib_ref] In silico, in vitro and in cellulo models for monitoring SARS-CoV-2 spike/human..., Lapaillerie [/bib_ref] to participate in important hydrogen bonds to the spike protein, also suggesting that the interaction of AB-00011778 with these residues could interfere with the ability of ACE2 to bind the spike protein.
The docking of AB-00047476 to ACE2 alone yielded a score of 78. [bib_ref] Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation, Wrapp [/bib_ref]. The most stable pose suggests the formation of one hydrogen bond with a length of 2.39 Å between Lys353 and the nitrogen from one of the benzothiazole groups. AB-00047476 and ACE2 also interact via p -p stacking interactions. These interactions are observed between His34 of ACE2 and a second benzothiazole group and between Phe72 and a third benzothiazole group, with lengths of 3.47 and 4.57 Å, respectively. Hydrophobic interactions between AB-00047476 and ACE2 Glu37, Leu39, and Phe72 are also well defined. For most of the docking results shown here, there are some interactions featuring residues from ACE2 and S-RBD that have been reported to form hydrogen bonds. In this case, the interactions between AB-00047476 and ACE2 Glu37 and Lys353 are particularly noteworthy. During the MD simulations of the S-RDB/ACE interface (6), Glu37 was shown to form a hydrogen bond with S-RDB Tyr505 for 56% of the simulation times, while Lys353 formed a hydrogen bond with Gly502 for 57% of the simulation times. Disruption of these interactions on the side of ACE2 could further help explain the inhibitory capabilities of this molecule.
Effects on the S-RBD/ACE2 association in vitro. To confirm that the selected drugs may physically prevent the formation of the S/ACE2 complex, we performed an AlphaLISA assay using the recombinant S minimal binding domain (RBD) fused to a 6ÂHis tag [S-RBD (His) [bib_ref] In silico, in vitro and in cellulo models for monitoring SARS-CoV-2 spike/human..., Lapaillerie [/bib_ref] ] and human ACE2 protein (ACE2-Biot). For this purpose, the AlphaLISA assay previously set up (6) and described in the legend to [fig_ref] FIG 7: Effects of AB-00011778 on SARS-CoV-2 Wuhan, alpha, delta, and omicron replication in... [/fig_ref] was adapted to 384-well microplates in a final reaction volume of 20 mL. As shown in [fig_ref] FIG 7: Effects of AB-00011778 on SARS-CoV-2 Wuhan, alpha, delta, and omicron replication in... [/fig_ref] and [fig_ref] FIG 4: Effects of AB-00011778 and AB-00047476 on the in vitro S-RBD/ACE2 interaction using... [/fig_ref] , the AlphaLISA data confirmed that both AB-00011778 and AB-00047476 could similarly interfere with the formation of the RBD/ACE2 complex, reaching 50% inhibition at 25 mM drug in both cases. A TruHits counterassay experiment did not show any significant effects from the compounds on the AslpaLISA (AS) signal, confirming the specific inhibition of the S-RBD/ACE2 interaction [fig_ref] FIG 7: Effects of AB-00011778 on SARS-CoV-2 Wuhan, alpha, delta, and omicron replication in... [/fig_ref]. To obtain additional information regarding the effects of the drugs on the kinetics of the S-RBD/ACE2 interaction, we used the biolayer interferometry (BLI) approach set up previously [bib_ref] In silico, in vitro and in cellulo models for monitoring SARS-CoV-2 spike/human..., Lapaillerie [/bib_ref]. In these competition binding assays, biotinylated ACE2 was loaded onto streptavidin biosensors and binding kinetics were carried out with 50 nM S-RBD(His) [bib_ref] In silico, in vitro and in cellulo models for monitoring SARS-CoV-2 spike/human..., Lapaillerie [/bib_ref] preincubated with increasing concentrations of compound AB-00047476 or AB-00011778 as described in Materials and Methods. Strong binding of S-RBD(His) [bib_ref] In silico, in vitro and in cellulo models for monitoring SARS-CoV-2 spike/human..., Lapaillerie [/bib_ref] to ACE2 was observed, as indicated by the wavelength shift in the BLI sensorgrams [fig_ref] FIG 4: Effects of AB-00011778 and AB-00047476 on the in vitro S-RBD/ACE2 interaction using... [/fig_ref] and C). The kinetic binding parameters (absorption rate constant [k a ] and dissociation constant [K d ]) and equilibrium dissociation constant (K D ) were determined after local fitting of the association and dissociation curves using a 1:1 binding model with ForteBio BLItz Pro 1.1 software. The K D was found to be 3.42 6 0.49 nM, confirming previous data [bib_ref] Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor, Lan [/bib_ref]. The addition of either AB-00011778 or AB-00047476 inhibited the interaction, leading to a strong increase in the K D to 111.0 6 16.0 nM and 68.8 6 5.1 nM, respectively [fig_ref] FIG 4: Effects of AB-00011778 and AB-00047476 on the in vitro S-RBD/ACE2 interaction using... [/fig_ref]. Determination of the kinetic constants in the absence or presence of drug showed that the compounds interfered with both the S-RBD/ACE2 association and dissociation steps, indicating that they are able to not only prevent the formation of the S-RBD/ACE2 complex but also decrease its stability, confirming the docking analyses.
Effects on the S-RBD/ACE2 association in cellulo. In a cellular context, both ACE2 and S are embedded in the membranous environment constituted by the viral envelope and the cellular membrane. Thus, we next investigated whether these compounds could also prevent the S/ACE2 interaction in a more physiological context. For this purpose, we used the cellular S-RBD/ACE2 interaction model developed previously [bib_ref] In silico, in vitro and in cellulo models for monitoring SARS-CoV-2 spike/human..., Lapaillerie [/bib_ref]. This model allows the association between recombinant S-RBD protein and the cellular membranous ACE2 protein to be monitored at the HEK293T-ACE2 cell surface by anti-S immunofluorescence [fig_ref] FIG 5: Effects of AB-00014778 and AB-00047476 on the S-RBD/ACE2 interaction in a cellular... [/fig_ref]. Flow cytometry analysis allowed the quantification of the global S-RBD association with the ACE2 protein [fig_ref] FIG 5: Effects of AB-00014778 and AB-00047476 on the S-RBD/ACE2 interaction in a cellular... [/fig_ref] , and competition with the soluble ACE2 protein confirmed the specificity of the interaction and validated the assay to test potential S/ ACE2 inhibitors [fig_ref] FIG 5: Effects of AB-00014778 and AB-00047476 on the S-RBD/ACE2 interaction in a cellular... [/fig_ref]. The addition of AB-00014778 or AB-00047476 significantly decreased the efficiency of the S-RBD/ACE2 association at the surface of the cell [fig_ref] FIG 5: Effects of AB-00014778 and AB-00047476 on the S-RBD/ACE2 interaction in a cellular... [/fig_ref]. AB-00014778 was found to be more efficient than AB-00047476, which is in agreement with data obtained from the previous infectivity and BLI assays reported in [fig_ref] FIG 2: Effect of AB-00047476 [/fig_ref]. Together, the data obtained demonstrate that the two selected drugs block the early interaction between S and ACE2, supporting, in concert with the data obtained with pseudoviruses, their effect on the early entry pathway mediated by this interaction.
Effect of the drugs on wild-type SARS-CoV-2 infectivity in naturally susceptible human cells. To complete the study, and prove the potency of the series, we next investigated whether this S/ACE2 inhibitory effect could sufficiently block the replication of wild-type circulating SARS-CoV-2 strains in different human cell lines naturally susceptible to infection.
The two best hits were tested in different pulmonary cell lines overexpressing, or not, the human ACE2 viral receptor after infection with the SARS-CoV-2 Wuhan reference strain. As shown in [fig_ref] FIG 6: Effects of AB-00011778 and AB-00047476 on SARS-CoV-2 replication in human pulmonary cells [/fig_ref] , both molecules induced inhibition of viral replication in A549-ACE2 cells, with 50% effective concentration (EC 50 ) values for AB-00011778 and AB-00047476 of 250 nM and 2.5 mM, respectively. The two molecules were then further tested in infection assays using the Calu3 human lung cancer cell line that naturally expresses the ACE2 receptor. As shown in [fig_ref] FIG 6: Effects of AB-00011778 and AB-00047476 on SARS-CoV-2 replication in human pulmonary cells [/fig_ref] , an inhibitory effect on viral replication was confirmed for both compounds, leading to 50% inhibition of viral replication at a concentration of approximatively 1 mM.
As reported in [fig_ref] TABLE 1: IC 50 , CC 50 , and SI values for AB-00047476 and... [/fig_ref] , based on the apparent 50% inhibitory concentration (IC 50 ) and 50% cytotoxic concentration (CC 50 ) values reported in the different cell lines, both compounds gave selectivity index (SI) values greater than 30, making them good candidates and anti-SARS-CoV-2-selective PPIs. AB-00011778 was found to be the most promising molecule, reaching an SI of .120 in A549-ACE2 cells.
## Chemical modulation of sars-cov-2 entry antimicrobial agents and chemotherapy
Effects of the selected drugs on SARS-CoV-2 circulating variant forms. To determine whether the selected drugs could be effective against the current major SARS-CoV-2 variants, we first performed in silico modeling and molecular dynamics simulations of the SARS-CoV-2 S/ACE2 complex with alpha, beta, gamma, and delta variants.
Models for the S-RDB/ACE2 complex formed with the different SARS-CoV-2 mutants were prepared starting from the X-ray structure 6M0J (resolution, 2.45 Å) (7), which represents the original SARS-CoV-2 spike protein receptor binding domain complexed with ACE2. Mutant variants were prepared by modeling the interface mutations using the mutagenesis feature in PyMOL 1.7.2.1 software, using the rotamer libraries available from Dunbrack [bib_ref] A smoothed backbone-dependent rotamer library for proteins derived from adaptive kernel density..., Shapovalov [/bib_ref]. Selection of the initial amino acid conformation of each mutant was based on the dominant conformer predicted, excluding clashes with other residues. This computational mutagenesis protocol has been previously used with success in the study of other enzymes (9, 10). The mutations modeled are reported in .
All four systems were subjected to the same molecular mechanics minimization and molecular dynamics procedure previously described, employing AMBER and the ff14sb force field. Molecular dynamics simulations were run for 400 ns for each mutant. Final trajectories were analyzed in terms of backbone root mean square deviation (RMSD), hydrogen bonds formed, and cluster analysis. From the cluster analysis performed on each mutant, a representative structure from the dominant cluster of conformations obtained for each mutant was selected using cpptraj from AmberTools and prepared for the molecular docking stage.
Protein-protein interaction inhibitors AB-00011778 and AB-00047476 were docked into the structures of each of the mutants using the PLP scoring function in GOLD with 500 independent genetic algorithm runs. Molecules were docked into (i) the S-RDB/ ACE2 interface and (ii) S-RDB alone. [fig_ref] TABLE 2: GOLD/PLP docking scores of AB-00011778 and AB-00047476 for the structures of the... [/fig_ref] presents the best scores obtained when docking the two molecules to the different variants, compared with the values obtained against the initial nonmutated S-RDB/ACE2 interface. For the omicron variant, the cluster analysis was performed on the 500-ns MD simulation trajectory performed by Lupala et al. [bib_ref] Mutations on RBD of SARS-CoV-2 Omicron variant result in stronger binding to..., Lupala [/bib_ref]. The results presented in [fig_ref] TABLE 2: GOLD/PLP docking scores of AB-00011778 and AB-00047476 for the structures of the... [/fig_ref] show that both molecules consistently bind with high docking scores to the different mutants, reinforcing the idea that these molecules can be equally effective in impairing the S/ACE2 association in the different mutants.
To confirm this, we tested the most promising drug (AB-00011778) on the four major circulating forms of SARS-CoV-2, namely, the original Wuhan strain and the alpha, delta, and omicron viruses in pulmonary A549-ACE2 cells. This compound blocked the viral replication of all variants with efficiency very similar to that of the Wuhan strain, leading to an IC 50 of 0.5 to 1 mM [fig_ref] FIG 7: Effects of AB-00011778 on SARS-CoV-2 Wuhan, alpha, delta, and omicron replication in... [/fig_ref].
Taken together, these data indicate that AB-00011778 is efficient against the main current SARS-CoV-2 strains and show an original entry inhibition mechanism by possibly acting simultaneously on distinct S/ACE2 interfaces. This compound thus constitutes a strong candidate for further evaluation in ACE2-humanized animal models and future clinical trials as a new SARS-CoV-2 entry inhibitor.
# Discussion
SARS-CoV-2 S/ACE2-mediated entry remains a major antiviral target not yet exploited in therapy, while the molecular mechanisms underlying this process are poorly characterized. By using in silico screening, we selected 10 hits from the French National Chemical Library based on their GOLD/PLP and AutoDock Vina scores. Among these 10 compounds, we identified two compounds showing potent entry inhibition in lentivirus-based pseudovirus infectivity assays. The specific inhibition of SARS-CoV-2 LV infectivity in comparison with VSV-g LVs suggests that the selected drugs target the early S/ACE2-dependent entry process reiterated in this model. The simplest explanation for the inhibition mechanism is blockage of the S/ACE2 association due to binding of the drug to the S/ACE2 interface. Biochemical approaches, including AlphaLISA and BLI, confirmed a direct effect of the drugs to block the S-RBD/ACE2 interaction by acting to prevent association and to dissociate the complex. Detailed analysis of the effects of the drugs on the kinetic parameters of S/ACE2 complex formation indicated that they could not only prevent the association between the two partners but also decrease the stability of the complex. These data are in full agreement with the molecular docking calculations performed on the S/ACE2 complex structure and on the S-RDB and ACE2 proteins alone, indicating that both drugs could make contact with the viral and cellular proteins. Interestingly, the molecular modeling studies reported in this work suggest that the selected drugs may interact with both the S and ACE2 proteins. This fully agrees with the BLI data indicating that the molecules could interfere with the formation of the S/ACE2 complex and decrease the stability of the formed complex. This may constitute an original inhibition mechanism of simultaneous blocking of various S/ACE2 interfaces, increasing the inhibition potency in addition to decreasing the emergence of resistance. This may also provide new tools to further study the functional implication of these interfaces during the viral entry stage. The two chosen compounds were also shown to inhibit the interaction between the soluble S-RBD and the ACE2 receptor expressed at the surface of HEK293T-ACE2 cells, confirming their capability to interfere with the formation of the S/ACE2 complex in a cell membrane context. To determine whether the selected drugs could be potential antiviral lead compounds, and to confirm the role of the S/ACE2 interfaces in the entry pathways, we tested them against wild-type viral replication using different cell lines, including naturally susceptible cells. The data indicated that both drugs were able to block, or reduce, the replication of the virus in pulmonary A549-ACE2 and Calu3 cells. While AB-00047476 was found to be less efficient in inhibiting viral replication in naturally susceptible pulmonary Calu3 cells, AB-00011778 maintained an efficient blocking effect, reaching 60 to 40% inhibition at 1 mM. Overall, both drugs showed IC 50 values between 0.25 and 5 mM among all the tested cell lines without showing significant cytotoxicity, allowing them to reach selectivity indices of .10. Notably, AB-00011778 showed the best inhibitory effects in all the cell lines, with SI values of .30 to 120. Further analysis of the inhibitory effects of AB-00011778 showed that it could also block the replication of the current major circulating forms (Wuhan, alpha, delta, and omicron) of SARS-CoV-2 in A549-ACE2-TMPRSS2 pulmonary cells. Interestingly, both ACE2 and TMPRSS2 were shown to dictate the viral entry [bib_ref] TMPRSS2 expression dictates the entry route used by SARS-CoV-2 to infect host..., Koch [/bib_ref]. Recent data from the literature showed that the expression of the cellular protease TMPRSS2 may dictate the entry pathway of the virus (5). The similar inhibition properties observed in A549-ACE2 and A549-ACE2-TMPRSS2 cells also indicate that the drug is able to block the different entry routes of the virus, underlining its broad antiviral properties. Taken together, these data identified AB-00011778 as a strong lead compound candidate for further development.
Aside from the recently licensed remdesivir and nirmatrelvir-ritonavir (Paxlovid), no effective drugs have been validated as potential specific anti-COVID treatments. Thus, the compounds reported here constitute the first molecules that act on the SARS-CoV-2 entry step by specifically targeting the S/ACE2 interfaces, making them very promising agents. A comparison of AB-00011778, remdesivir, and ritonavir (one of the two antiproteases included in Paxlovid) showed that while the remdesivir remains the most efficient molecule in our assay, AB-00011778 was more efficient than the ritonavir molecule in inhibiting the SARS-CoV-2 replication in human pulmonary A549-ACE2 cells (see in the supplemental material). Of note, in contrast to the repurposed remdesivir and ritonavir drugs acting on different replication steps, AB-00011778 is an original molecule that has been specifically selected to block SARS-CoV-2 entry and has not yet been optimized. The low cytotoxicity observed for this molecule in various cell lines also paves the way for future animal model studies and clinical trials to fully evaluate their antiviral properties and therapeutic potency. Our work also provides a strong molecular and structural basis for further structure/function relationship studies to design potential optimized derivatives from the initially selected hits currently developed in our laboratory. Furthermore, these studies will help in the further characterization of the molecular mechanism underlying the inhibition of the S/ACE2 interaction by the selected drugs and will certainly allow optimization of this original antiviral process, leading to new promising agents usable for COVID-19 curative approaches.
# Materials and methods
In silico molecular dynamics simulation of the SARS-CoV-2 S/ACE2 complex and virtual screening. A model was prepared considering the spike protein S1 of SARS-CoV-2 complexed with human ACE2 starting from the X-ray structure 6M0J (resolution, 2.45 Å) [bib_ref] Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor, Lan [/bib_ref]. This structure contains the receptor binding domain (RBD) of S1 and includes all the amino acid residues of the RBD, with well-defined density between the residues on S1 RDB and ACE2. The model was treated with PropKa (12) to assign protonation states and subjected to molecular dynamics simulations for 400 ns based on the AMBER ff14SB force field as described in detail previously [bib_ref] In silico, in vitro and in cellulo models for monitoring SARS-CoV-2 spike/human..., Lapaillerie [/bib_ref]. Final trajectories were analyzed in terms of backbone root mean square deviation (RMSD), hydrogen bonds formed, and cluster analysis. From the cluster analysis performed on the ensemble of poses generated from the MD simulations, one representative structure from each of the 8 dominant clusters was selected and analyzed with FPocket software [bib_ref] Fpocket: an open source platform for ligand pocket detection, Guilloux [/bib_ref]. This approached enabled the identification of possible druggable pockets in the interfacial S1-ACE2 region in each of the selected structures and in the initial model prepared from the X-ray structure. These were described in detail previously [bib_ref] In silico, in vitro and in cellulo models for monitoring SARS-CoV-2 spike/human..., Lapaillerie [/bib_ref].
Structure-based virtual screening was performed using the following four molecular libraries: (i) the French National Chemical Library (Chimiothèque Nationale) (70,000 molecules; https://chembiofrance.cn .cnrs.fr/fr/composante/chimiotheque), (ii) the Mu.Ta.Lig. Virtual Chemotheca (ca. 60,000 molecules; http://chemotheca.unicz.it), (iii) the Inhibitors of Protein-Protein Interactions Database (1,956 molecules; https://ippidb.pasteur.fr), and (iv) the ZINC FDA Approved and ZINC In-Trials Database (1,379 and 5,811 molecules for evaluation for drug repurposing; https://zinc.docking.org). These libraries were screened against the SARS-CoV-2 S/ACE2 complex (model 1) [fig_ref] FIG 1: Selection of drugs targeting the S/ACE2 interaction by virtual screening studies [/fig_ref] and against SARS-CoV-2 S alone (model 2) [fig_ref] FIG 1: Selection of drugs targeting the S/ACE2 interaction by virtual screening studies [/fig_ref] , with two independent docking software programs and scoring functions: AutoDock Vina [bib_ref] AutoDock Vina: improving the speed and accuracy of docking with a new..., Trott [/bib_ref] and GOLD (PLP scoring function) [bib_ref] Empirical scoring functions for advanced protein-ligand docking with PLANTS, Korb [/bib_ref]. It should be noted that the different docking program results have different values and scales. The score for PLP is dimensionless, and the higher the score, the better the binding affinity. On the other hand, AutoDock Vina uses a metric that resembles binding free energy (in kcal/mol), so a more negative value suggests a stronger affinity.
Compounds were sorted based on their Vina and PLP scores against model 1 and model 2. A selection of 20 compounds based on the most appealing compounds from each library was then made. This selection took into consideration not only the score against both models with Vina and/or PLP but also the chemical diversity among the molecules selected and the physicochemical properties of the molecules [fig_ref] TABLE 1: IC 50 , CC 50 , and SI values for AB-00047476 and... [/fig_ref] and [fig_ref] FIG 2: Effect of AB-00047476 [/fig_ref].
Molecular docking of the LEAD compounds. For the molecular docking of AB-00011778 and AB-00047476, the PLP scoring function from GOLD was used with 500 independent genetic algorithm runs. Molecules were docked into (i) the S-RDB/ACE2 interface, (ii) S-RDB alone, and (iii) ACE2 alone. For (ii) and (iii), the cavity for docking was defined based on the known binding region of the other partner in the complex formed (i.e., of ACE2 for the S-RDB model [ii] and S-RDB for the ACE2 model [iii]) plus a radius of 10 Å. The same procedure was later extended to AB-00011778_2 and AB-00011778_6. Analysis of the interactions was done with the help of the protein-ligand interaction profiler [bib_ref] PLIP 2021: expanding the scope of the protein-ligand interaction profiler to DNA..., Adasme [/bib_ref].
Chemicals. Bis-indolyl pyridines and their derivatives have been synthetized as previously reported [bib_ref] Synthesis of 3,5-bis(2-indolyl)pyridine and 3-[(2-indolyl)-5-phenyl]pyridine derivatives as CDK inhibitors and cytotoxic agents, Jacquemard [/bib_ref]. The tris-benzothiazole triphenylamine derivative AB-00047476 belongs to the Institut Curie/CNRS chemical library (purity of .95% by proton 1 H nuclear magnetic resonance [NMR] spectrometry), and its chemical synthesis was previously described [bib_ref] meta-Substituted triphenylamines as new dyes displaying exceptionally large Stokes shifts, Bordeau [/bib_ref]. All the drugs had been prediluted in 100% dimethyl sulfoxide (DMSO), and the same volume of each serial predilution was added in the assays in order to keep the same DMSO concentrations and to avoid precipitation of the drug.
Proteins and antibodies. SARS-CoV-2 (438-516) S-RBD(His) [bib_ref] In silico, in vitro and in cellulo models for monitoring SARS-CoV-2 spike/human..., Lapaillerie [/bib_ref] , including the minimal receptor binding motif previously described [bib_ref] Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation, Wrapp [/bib_ref] , and biotinylated human ACE2 were purchased from Fisher Scientific (reference no. 16534204 and 16545164, respectively). Monoclonal anti-6ÂHis tag antibody was purchased from Abcam (reference no. ab18184; dilution, 1/200). Anti-a-tubulin antibody was purchased from Sigma (reference no. T6199; dilution, 1/500). Secondary goat anti-mouse antibody coupled to Alexa Fluor 488 was purchased from Fisher Scientific (reference no. Allo2g; dilution, 1/400).
AlphaLISA binding assays. The AlphaLISA assay development was performed as previously described (5) using the recombinant S minimal binding domain (RBD) fused to a 6ÂHis tag [S-RBD(His) [bib_ref] In silico, in vitro and in cellulo models for monitoring SARS-CoV-2 spike/human..., Lapaillerie [/bib_ref] ] and biotinylated human ACE2 protein (ACE2-Biot). The binding conditions were adapted for use in 384well plates (white OptiPlate; reference no. 6007290; PerkinElmer, Waltham, MA) in a final reaction volume of 20 mL and are fully described in the legend to [fig_ref] FIG 7: Effects of AB-00011778 on SARS-CoV-2 Wuhan, alpha, delta, and omicron replication in... [/fig_ref]. Data were analyzed with GraphPad Prism 5.01 software. The AlphaLISA binding data obtained with the compounds were compared to the 1% DMSO control condition. For hit validation, a TruHits counterassay kit (reference no. AL900D; PerkinElmer) was used.
BLI binding assays. Biolayer interferometry (BLI) experiments were performed on a BLItz instrument (Sartorius, Göttingen, Germany) to measure the binding of S-RBD(His) [bib_ref] In silico, in vitro and in cellulo models for monitoring SARS-CoV-2 spike/human..., Lapaillerie [/bib_ref] to ACE2-Biot. The BLI assay was set up as previously described [bib_ref] TMPRSS2 expression dictates the entry route used by SARS-CoV-2 to infect host..., Koch [/bib_ref] with some adaptations for the competition binding analysis with drugs. All sample dilutions and baseline steps were carried out using the same reaction buffer (phosphate-buffered saline, pH 7.4, 0.1% [wt/vol] bovine serum albumin, 1% [vol/vol] DMSO). Streptavidin biosensors (Sartorius; reference no. 18-5019) were first prewetted for 10 min with the reaction buffer, and 1 mM ACE2-Biot was then loaded onto the coated biosensors for 500 s in order to reach a binding value of ;3 nm. For the competition kinetic analyses, protein samples were prepared at a concentration of 50 nM S-RBD(His) [bib_ref] In silico, in vitro and in cellulo models for monitoring SARS-CoV-2 spike/human..., Lapaillerie [/bib_ref] in accordance with our previous BLI binding studies and were preincubated for 15 min at room temperature with increased concentrations (0 to 25 mM) of AB-00047476 or AB-00011778 drugs. Binding kinetics were divided into three steps. First, the baseline with the reaction buffer was measured for 60 s. For the association step, each loaded biosensor was dipped into S-RBD sample with drugs or DMSO as a control for 300 s with a 2,200-rpm shaking speed. Finally, the dissociation step of the bound S-RBD(His) [bib_ref] In silico, in vitro and in cellulo models for monitoring SARS-CoV-2 spike/human..., Lapaillerie [/bib_ref] was monitored by dipping the biosensor back into the reaction buffer for 300 s. Systematic baseline drift correction was done by subtracting the shift recorded for the biosensor loaded with ACE2 but incubated with reaction buffer. The association and dissociation experimental curves were locally fitted using a 1:1 binding model with the BLItz Pro 1.1 software, and the kinetic binding parameters (k on , k off ) were determined as the means of results of two to three independent experiments. The equilibrium dissociation constant (K D ) was calculated as K D = k off /k on . Sensorgram curves were plotted using Prism 5.0 software (GraphPad Software, La Jolla, CA).
Cells and lentiviral vector production. Lentivirus vector production was done by the service platform Vect'UB, (INSERM US 005-CNRS UMS 3427-TBM-Core, Université de Bordeaux, France). Lentiviral particles were produced by transient transfection of HEK293T (human embryonic kidney) cells according to standard protocols. In brief, subconfluent HEK293T cells were cotransfected with a lentiviral genome (psPAX2) (gift from Didier Trono (Addgene plasmid no. 12260)), with an envelope coding plasmid (pMD2G-VSVG or wild-type SARS-CoV-2 spike protein [HDM_IDTSpike_fixK; kind gift from Bloom's laboratory] [bib_ref] Protocol and reagents for pseudotyping lentiviral particles with SARS-CoV-2 spike protein for..., Crawford [/bib_ref] , and with vector constructs (44: pRRLSIN-PPT-hPGK-eGFP-WPRE or pHAGE_EF1alnt_ACE2_WT) by calcium phosphate precipitation. LVs were harvested 48 h posttransfection and concentrated by ultracentrifugation. Viral titers of VSV-g pseudotype pLV lentivectors were determined by transducing HEK293T cells with serial dilutions of viral supernatant, and eGFP expression was quantified 5 days later by flow cytometry analysis. For SARS-CoV-2 spike pseudotype, p24 antigen levels were measured in the concentrated viral supernatants by an enzyme-linked immunosorbent assay (Innotest HIV Ag nAb; Fugibio, France) and viral titers were estimated by comparing p24 antigen levels of each lentiviral supernatant with a similar VSV-g pseudotype lentiviral supernatant produced simultaneously.
HEK293T-ACE2 cell lines were generated by lentiviral transduction using pHAGE_EF1alnt_ACE2_WT plasmid (kind gift from Bloom's laboratory [bib_ref] Protocol and reagents for pseudotyping lentiviral particles with SARS-CoV-2 spike protein for..., Crawford [/bib_ref]. HEK293T cells (200,000 cells) were then transduced with an optimized concentration of ACE2 lentiviral particles in 6-well plates. The efficacy of transduction was assessed using real-time PCR 10 days postinfection. ACE2 provirus DNA from this cell line was quantified by quantitative PCR (qPCR) using the DDC T method in comparison with a 1-copy cell line. DNA from two different cell clones (human 293T and K562 cells), containing a single integrated copy of the provirus, was used as a normalized cell line. HEK293T cells were cultured in Dulbecco modified Eagle medium (DMEM) supplemented with 1% penicillin-streptomycin and 10% fetal bovine serum (FBS).
A549 cells (human alveolar basal epithelial carcinoma), Calu3 (human bronchial epithelial carcinoma), and Vero E6 cells (African green monkey kidney cells) were maintained in DMEM (Invitrogen Life Technologies) supplemented with 10% heat-inactivated FBS, 1% penicillin/streptomycin (P/S), and 1% GlutaMAX (Life Technologies).
The MTT cell viability assay was performed in accordance with the manufacturer's protocols and is further described in the legend to [fig_ref] FIG 5: Effects of AB-00014778 and AB-00047476 on the S-RBD/ACE2 interaction in a cellular... [/fig_ref].
In cellulo imaging of the SARS-CoV-2 S-RBD/ACE2 interaction. A total of 10,000 HEK293T and HEK293T-ACE2 cells were plated on glass coverslips pretreated with poly-L-lysine solution, 0.01% (Sigma reference no. P4832), for 5 min at room temperature. After 24 h, the RBD recombinant protein (4 to 40 pmol) was added to the cells in 100 mL phosphate-buffered saline (PBS). The cells were then washed and fixed (paraformaldehyde [PFA], 4%) at different time points. Cell imaging was performed as described above.
For quantification of the S-RBD/ACE2 interaction in a cellular context, 45,000 HEK293T and HEK293T-ACE2 cells were incubated for 45 min at 37°C in 100 mL DMEM and increasing concentrations of RBD recombinant protein. One milliliter of PBS was added, and the cells were centrifuged for 5 min at 2,500 rpm. Fifty microliters of anti-His antibody at 1/200 in DMEM was added, and the cells were incubated for a further 45 min at 37°C. After PBS washes, 50 mL of a 1/400 DMEM solution of secondary antibody was added and the cells were incubated for 30 min at 37°C. After PBS washes, the cells were resuspended in 200 mL PBS-2% FBS-2 mM EDTA and the percentage of fluorescein isothiocyanate (FITC)-positive cells was quantified by flow cytometry.
SARS-CoV-2 production and infection assays. SARS-CoV-2 strain 220_95 (EPI_ISL_469284) was isolated from nasopharyngeal swab specimens collected at Service de Virologie (Hospital Saint Louis, Paris, France) and grown as previously described [bib_ref] SARS-CoV-2 induces human plasmacytoid predendritic cell diversification via UNC93B and IRAK4, Onodi [/bib_ref]. Briefly, virus was propagated on Vero E6 cells in DMEM-2% (DMEM supplemented with 2% FBS, 1% P/S, 1% GlutaMAX, and 25 mM HEPES). Viruses were purified through a 20% sucrose cushion by ultracentrifugation at 80,000 Â g for 2 h at 4°C. Pellets were resuspended in 1Â HNE (25 mM HEPES, 100 mM NaCl, 0.5 mM EDTA), aliquoted, and stored at 280°C.
[fig] FIG 1: Selection of drugs targeting the S/ACE2 interaction by virtual screening studies. (A and B) The virtual screenings targeting the reported region in the S/ACE2 complex and in S alone were performed by molecular dynamics calculations. The surface representation corresponds to S, while the ribbon representations represent ACE2. (C and D) Results of the virtual drug screening. [/fig]
[fig] FIG 2: Effect of AB-00047476 (A) and AB-00011778 (B) on SARS-CoV-2 and VSV-g lentivirus infectivity. AB-00047476 and AB-00011778 were selected from 10 mM concentrations from a previous screen for further characterization using increasing concentrations of drug. Data are shown as the means of results of at least three independent experiments 6 standard deviations. ***, P , 0.001; **, P , 0.01 (Student's t test). [/fig]
[fig] FIG 3: Molecular docking of AB-00011778 and AB-00047476 to the spike/ACE2 complex and to the spike protein alone. Data were obtained from the predicted binding poses of the compounds to the S-RBD and to the S-RBD/ACE2 interface. The interacting amino acid residues are highlighted. (A) AB-00011778 bound to the spike/ACE2 complex; (B) AB-00047476 bound to the spike/ACE2 complex; (C) AB-00011778 bound to the spike protein alone; (D) AB-00047476 bound to the spike protein alone. [/fig]
[fig] FIG 4: Effects of AB-00011778 and AB-00047476 on the in vitro S-RBD/ACE2 interaction using AlphaLISA and biolayer interferometry (BLI) technologies. (A) SARS-CoV-2 S-RBD(His)6 and human ACE2-Biot interactions were first monitored by AlphaLISA using a 3 nM concentration of each protein. Increasing concentrations of AB-00011778 and AB-00047476 were incubated for 30 min with S-RBD(His) 6 before being mixed with ACE2-Biot for 2 h. The microplate results were read after 2 h of incubation with the anti-6ÂHis acceptor and streptavidin donor beads. Data obtained with the compounds were compared to the DMSO control and are reported as the mean percentage of binding inhibition from two or three independent experiments in duplicate 6 standard deviation. (B and C) The effect of the molecules on the binding kinetics of S-RBD to biotinylated ACE2 immobilized on a streptavidin biosensor was also determined using BLI experiments. Baseline reaction buffer was measured for 60 s. For the association step (300 s), each loaded biosensor was dipped into 50 nM S-RBD(His) 6 preincubated for 15 min with increasing concentrations of AB-00011778 or AB-00047476. The dissociation step was subsequently measured for 300 s. Sensorgram curves shown in panels B and C were plotted using Prism 5.0 software (GraphPad Software, La Jolla, CA). The association and dissociation experimental curves were locally fitted using a 1:1 binding model with BLItz Pro 1.1 software. (D) Kinetic binding parameters (k on , k off ) and the equilibrium dissociation constant (K D ) were determined as the means of results of two to three independent experiments. Data are shown as the means of results of two to three independent experiments 6 standard deviations. [/fig]
[fig] FIG 5: Effects of AB-00014778 and AB-00047476 on the S-RBD/ACE2 interaction in a cellular context. The SARS-CoV-2 S-RBD fragment was added to either HEK293T or HEK293-ACE2 cells for 0 to 60 min, and immunofluorescence staining was performed using an anti-6ÂHis antibody and secondary antibody coupled to Alexa Fluor 488. (A) Cells were observed by epifluorescence microscopy. (B) Cells were also analyzed using flow cytometry to detect the percentage of FITC-positive cells over time. (C and D) Increasing concentrations of either soluble ACE2 (C) or drugs (D) were added to the RBD. Data are reported as the mean percentage of positive cells from three to five independent experiments 6 standard deviation. **, P , 0.05; ***, P , 0.005 (Student's t test). [/fig]
[fig] FIG 6: Effects of AB-00011778 and AB-00047476 on SARS-CoV-2 replication in human pulmonary cells. A549-ACE2 (A) or Calu3 (B) cells were incubated with the SARS-CoV-2 reference strain (MOI = 1) and increasing concentrations of the tested compounds. Replication was evaluated by quantifying the viral genome copies after 24 h. Data are reported as the means of results from five independent experiments and are expressed as a percentage of the control without molecule 6 standard deviation. ***, P , 0.001; **, P , 0.01; *, P , 0.05 (Student's t test). [/fig]
[fig] FIG 7: Effects of AB-00011778 on SARS-CoV-2 Wuhan, alpha, delta, and omicron replication in human pulmonary cells. A549-ACE2-TMPRSS2 cells were incubated with the SARS-CoV-2 Wuhan, alpha, or delta strain (MOI = 1) and increasing concentrations of drugs. (A and B) Replication was evaluated by quantifying the viral gene E (A) and S (B) genome copies normalized to actin RNA after 24 h (results of a representative experiment are shown here). (C) Means of results from two to three independent experiments expressed as a percentage of the control without molecule 6 standard deviation. [/fig]
[table] TABLE 1: IC 50 , CC 50 , and SI values for AB-00047476 and AB-00011778 a CC 50 was calculated using four-parameter variable slope sigmoidal dose-response models based on MTT assays. [/table]
[table] TABLE 2: GOLD/PLP docking scores of AB-00011778 and AB-00047476 for the structures of the different SARS-CoV-2 mutants [/table]
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The role of maternal health care services as predictors of time to modern contraceptive use after childbirth in Northwest Ethiopia: Application of the shared frailty survival analysis
OPEN ACCESSCitation: Emiru AA, Alene GD, Debelew GT (2020) The role of maternal health care services as predictors of time to modern contraceptive use after childbirth in Northwest Ethiopia: Application of the shared frailty survival analysis. PLoS ONE 15 (2): e0228678. https://doi.org/10.A substantial proportion of women did not use modern contraceptive methods in the first year after birth. Maternal services were found to be the sole predictors in postpartum contraceptive use. Findings suggest the importance of linking postpartum family planning along the continuum of care. The observed heterogeneity at cluster level also urges the need of disaggregating data for decision-making.IntroductionAt the turn of the 21 st century maternal mortality remains a major public health challenge for many developing countries, and nowhere are global inequalities more starkly clear than in maternal death[1]. The low-income countries accounted for 99% of these deaths of which twothird occurs in Sub-Saharan Africa where Ethiopia lies[1,2]. At 412 deaths per 100,000 live births[3], Ethiopia's maternal mortality ratio is one of the highest even by the standard of developing countries[4].However, it is known that many of the maternal deaths can be prevented with appropriate maternal care during pregnancy, delivery and post natal periods[5,6]. With this in view, women access health services more often during the time of pregnancy, delivery, and the first year after birth than other periods[7].These points of contact, whether part of a routine or emergency care, are opportunities for providers to screen for, counsel, and hence to address the contraceptive needs of postpartum women and couples[8]. Therefore, promoting contraceptive use during each contact in the continuum is considered an important strategy for addressing the widespread unmet needs in family planning[9].Despite this, the vast majority of postpartum women in developing countries missed the opportunities at each points of contacts[8,10]. With this, postpartum women are among those with the greatest unmet need for family planning than other periods [6, 10, 11]. In Ethiopia, unmet need for family planning in the postpartum period is also much higher than women outside of the extended postpartum period; while 16.2% women, in general, do have unmet need for family planning[12], the unmet need in the first year after childbirth reaches as high as 44 percent[13].Women's decision to use modern contraceptives in the first year of postpartum is influenced by a complex array of factors within the community and health system[14,15]. Postpartum women often do not realize that they are at risk of pregnancy when they are amenorrheic or breastfeeding[15]. Postpartum contraception adoption has also a social connotation; the postpartum period is the time during which a woman needs to adjust herself to new roles to care for her newborn[16]. Furthermore, male dominance and subordination of women, as well as mistaken beliefs, and religious faith of the population are other impediments to the acceptance of contraception in developing countries, including Ethiopia[15].Beyond the socio-cultural barriers, insufficient contraceptive method mixes, and poor family planning service integration with other health services in many settings also appear to be formidable obstacles remaining to be correct[8,17].Several studies have been carried out on postpartum contraceptive use in the study area in particular and Ethiopia at large[18][19][20]; yet, most of the earlier studies, if any, have ignored the hierarchical facts. It is, however, evidenced that clustering (frailty) has an effect on modeling the predictors of time to contraception[21]. Consequently, modeling time to contraceptive Predictors of time to contraceptive use after birth PLOS ONE | https://doi.org/10.
# Introduction
The first year after birth is an ideal time to offer contraception services, as many women have many opportunities to be in contact with the health care system. Nevertheless, a large number of postpartum women in developing countries do not use the service owing to the interplay of factors operating at various stages. Therefore, this study aimed to assess predictors of modern contraceptive use in the extended postpartum period.
# Methods
A community based retrospective cross-sectional study was done among 1281 women who gave birth within 12 months preceding the survey. Kaplan-Meier plots and log rank tests were used to explore the rate of modern contraceptive use. The Weibull regression survival model with multivariate frailty was employed to identify the predictors of time to contraception.
# Results
Of the respondents, 59.1% (95% CI: 56.8%-62.2%) had started using modern contraceptive methods within 12 months after birth. By the second month after birth, only 11.1 percent of the women surveyed started to use a contraceptive method, which increased steadily to 25.9%, 37.7%, and 59.5% at 6, 9, and 12 months, respectively. The most preferred contraceptive method was injectable (71.5%), followed by implants (21.5%). Women's education (aHR = 1.29; 95%CI: 1.02, 1.66), four or more antenatal care (aHR = 1.59; 95% CI: 1.22, 2.06), early initiation of antenatal care (aHR = 2.03; 95% CI: 1.28, 3.21), and early postnatal checkup (aHR = 1.39; 95% CI: 1.12, 1.73) were statistically significant predictors of earlier initiation of modern contraceptive methods. use ignoring the frailty terms may lead to biased estimates of parameters and their respective standard errors [bib_ref] Parametric frailty and shared frailty survival models, Gutierrez [/bib_ref]. Furthermore, according to the current Ethiopian Demographic and health survey (EDHS), regional disparities in contraceptive adoption have been reported, with higher rates observed in the Amhara region(where the study area is found) and Addis Ababa(the capital city of the country) than the rest of the regions. Nevertheless, this was a survey report rather than an empirical study, and the contribution of explanatory factors was not examined.
In this regard, addressing these gaps would have significant implications for policymakers, health planners, and clinicians. Therefore, considering the hierarchal nature of our data we attempted to provide an understanding of factors associated with the timing of postpartum contraceptive use using a shared frailty survival model.
# Materials and methods
## Study setting
The study was carried out in West Gojjam zone, which is one of the eleven zones found in the Amhara region of Ethiopia. Administratively, the zone is subdivided into 13 rural districts and 2 town administrations with a projected total population of 2,611,925 ,017 rural and 417,908 urban) people. The number of females in the reproductive age group was 615,892, accounting for 23.58% of the total population.
The zone had over 598 health facilities (6 public primary hospitals, 103 health centers, 374 functional health posts, 114 private clinics, and 1 private hospital) at the time of the survey. Family planning, antenatal care, labor and delivery, and postnatal services are provided free of charge in all the public health facilities.
## Study design and population
A community-based retrospective cross-sectional study was conducted on reproductive-aged women whose most recent birth was within 12months preceding the survey.
## Sample size and sampling procedure
The required sample size was done through the STAT CALC program of the Epi-Info statistical package V.7.0. This study was part of a large study done on the continuum of maternal health care with multiple objectives. For each objective alternative sample sizes were computed considering both the double and single population formulas; the detail of the sample size calculation and sampling procedure is publicly posted in the research square. Of the alternative sample sizes computed based on different indicators, the largest sample size (1294 women) was obtained when considering the following assumptions; 95% confidence level, 4%margin of error, 16.5% proportion of PNC utilization, design effect of 2, and 10% non-response rate. However, during the time of data collection 1337 women who met the inclusion criteria were included in this study to increase the power of the study.
A multistage sampling technique was used to identify the study participants. First, five out of fifteen districts in the Zone (four rural districts and one town administration) were selected using simple random sampling. Second, thirteen kebeles (the smallest administrative units in Ethiopia) were chosen randomly by taking in to account the number of kebeles in each district. Then, a complete list of deliveries that took place within 12 months before the survey was identified from the family folder of health extension workers in the respective kebeles. Finally, 1337 eligible women who met the inclusion criteria were selected.
## Study variables and measurement
The outcome variable was modern contraceptive use within 12 months following the last birth. A woman who started using modern contraceptives was coded as "1", and otherwise "0".
The explanatory variables included: socioeconomic variables (such as place of residence, maternal/paternal education, household wealth index, and primary Health care (PHC) facilities per 25,000 populations at district level); demographic characteristics (age of women, occupation of women and husbands); and reproductive variables (such as desirability of the pregnancy, family size, birth interval, number and timing of antenatal visits, mode of delivery, and postnatal care.
The wealth index was generated from the household's cumulative living standard based on ownership of specified assets using factor analysis and was later categorized into terciles (poor, middle and rich).
The two quantitative terms, survivor function S (t) and hazard function h (t), are important in any survival analysis. In relation to our study, the survivor function is the probability that a postpartum woman "survives" longer than some specified time "t" without started taking modern contraceptive methods after childbirth. Whereas, the hazard function gives the instantaneous potential per unit time to start using modern contraception after time "t", given that the woman had not started taking any modern contraceptive up to time "t".
## Data collection process
The household data were collected using a pre-tested interviewer-administered questionnaire, developed in the local language, Amharic. Fifteen nurses and five public health officers were deployed as data collectors and supervisors, respectively after receiving two days of intensive training. Data regarding socio-economic, demographic, and reproductive characteristics were collected among women who gave birth (either at home or in a health facility) to a baby within 12 months before the survey. Besides, the number of PHC providing maternity and reproductive health services per total population was assessed at the district level, and the result had been linked to the individual woman in the corresponding household survey.
## Data processing and analysis
The analysis was done using STATA 14.0. Both descriptive statistics and survival analysis techniques were used in analyzing the data. First, an assessment of the time-to-modern contraceptive use after birth was done using life tables based on the Kaplan-Meier (K-M) estimate. Second, the Log-rank Chi-square test was used to examine the differences in the survival curves for different categories of each study variable. Third, the multivariate (or shared frailty) survival analysis was done by assuming different parametric distributions for the baseline hazard function and using gamma for frailty distributions.
The shared frailty approach is a conditional independence model for time to event data, where the frailty term (the random effect) is common to all subjects in a cluster. In our study women who were living in the same cluster (kebele) were more likely to have outcomes (post-partum contraceptive use) that are correlated with one another, and, thus independence between event times cannot be assumed. Moreover, it is unlikely to include all the relevant covariates in the model. With that in mind, using the cox proportional hazard model could not account for all the variability in the observed failure times. Therefore, it was reasonable to apply the shared frailty survival model, that accounts for heterogeneity caused by unmeasured covariates, as an alternative to the standard cox survival model [bib_ref] Parametric frailty and shared frailty survival models, Gutierrez [/bib_ref]. The conditional hazard function for the Weibull shared frailty survival model used in this study is defined as:
[formula] h ij ðt=z i Þ ¼ z i rt rÀ 1 expðb 0 X ij Þ; i ¼ 1; . . . ; n; j ¼ 1; . . . ; k i [/formula]
Where i indicates the i th cluster (kebele), j indicates the j th individual in the i th cluster, pt p−1 is the baseline hazard, X ij is the vector of covariates for subject j in cluster i, β' is a vector of regression coefficients, and Z i is the frailty term. In this study the frailty Z i was supposed to follow a gamma distribution g(z; θ, θ), which is the most common and widely used distribution for determining the frailty effect [bib_ref] Parametric frailty and shared frailty survival models, Gutierrez [/bib_ref].
The Akaike Information Criterion (AIC) was used to select the appropriate model, whilst the Cox-Snell residual plot analysis was done to evaluate the overall model fitness. Furthermore, interaction between the independent variables for contraceptive use was tested. Finally, the frailty effects, Kendall's Tau, and hazard ratio at 95% confidence interval were estimated for the selected model.
# Ethical approval
This study was approved by the Research Ethical review committee of the College of Medicine and Health Sciences, Bahir Dar University (reference number: 087/18-04). Letters of permission were secured from the Amhara Regional State Health Bureau and respective health offices. Also, oral informed consent was received from each study participant. The data obtained from each study participants was kept confidential throughout the process of study, and the name of the participants was replaced by code.
# Results
# Background characteristics
A total of 1281 reproductive-age women participated in this study giving a response rate of 95.8%. More than half 674(52.6%) of the women were in the age group of 25-34 years with a mean (± SD) age of 30.3(±6.0) years. Little more than three-quarters, 978(76.3%), of the sampled women were rural residents, and 862(67.3%) of them belonged to the lower two wealth quintiles.
Concerning the reproductive characteristics, 511(39.9%) of them had at least 4 ANC visits, 194(15.1%) had their first ANC visit within the first four months after conception. Also, the highest proportion, 672(52.5%) of women had deliveries at home and almost a similar proportion of 719 (56.1%) women did not have any health check within the six weeks of postpartum [fig_ref] Table 1: Background characteristics of postpartum women in West Gojjam Zone, Northwest Ethiopia, 2018 [/fig_ref].
## Results from survival analysis
Among all the respondents, 59.5% (95% CI: 56.8%-62.2%) had started using any modern contraceptive method after the last birth while the remaining were right-censored as of the time of the survey. Contraceptive users have contributed 11,737 months (978 women-years) of follow up, with an average follow-up time of 9.16 (95% CI = 8.96-9.37) months [fig_ref] Fig 1: Kaplan Meier survival function curve showing time to modern contraceptive use after... [/fig_ref].
Our findings revealed that only 11.1 percent of the postpartum women surveyed started to use a contraceptive method by the second month after childbirth. The proportion of users then increased steadily over the months reaching 25.9%, 37.7%, and 59.5% at 6, 9, and 12 months, respectively.
The illustrations in [fig_ref] Fig 2: Kaplan Meier estimate curve of postpartum family planning use within 12 months... [/fig_ref] provide insights into the features of the differences in the Kaplan-Meier survival curves by selected maternal characteristics. Clearly, the overall estimated survivor function showed that mothers started taking modern contraception after the
2 nd month of the last birth. It is also evident that the survival curves are substantially different among women whose first antenatal visit was during the first trimester, during the second trimester, or started in the third trimester. Similarly, the curves differ for various educational categories of women, the number of antenatal follow-ups, place of residence, and history of menstrual resumption after birth during the first12 months following the last childbirth [fig_ref] Fig 2: Kaplan Meier estimate curve of postpartum family planning use within 12 months... [/fig_ref].
Before we fit the final model, the observed difference in survival experiences in different groups was also assessed using the log-rank test. The variables considered include maternal age, maternal and paternal educational attainment, frequency and timing of first ANC follow up, type of delivery, postnatal care within the first two days after birth, PHC to population ratio, family size, and household wealth index. The log-rank test result revealed that each of the covariates, except for facility to population ratio, has a significant Wald test when using α = 20%. However, we kept facility to population ratio in the final model as access to the healthcare facility was considered an important variable in different literature. Moreover, the sample size is sufficient to accommodate more predictors.
Then, all the covariates that were selected in the log-rank test at a 20% level of significance were fitted in the parametric shared frailty models of exponential, Weibull, log-logistic, and log-normal distributions by using cluster (kebele) as frailty term. The shared frailty model with the Weibull baseline hazard function had the smallest AIC value [fig_ref] Table 2: Comparison of fitness of different parametric frailty models based on the Akaike... [/fig_ref] than the other frailty models, and hence was selected to describe time-to-postpartum contraception data. The AIC values for all the parametric frailty models are summarized in [fig_ref] Table 2: Comparison of fitness of different parametric frailty models based on the Akaike... [/fig_ref] below. Tests of Unobserved Heterogeneity. The effect of clustering (unobserved heterogeneity) between the clusters (Kebeles) was tested using the likelihood ratio test (LRT). The results of this test revealed that the variance of the random effect was significantly greater than zero (θ = 0.06; p value< 0.05) in the weibull baseline frailty model, suggesting the unescapable role of the unmeasured cluster effects in the model. In addition, Kendall's tau (τ) value of 0.03, suggests a positive correlation between times to contraception within the clusters [fig_ref] Table 3: Results of Multivariable analysis of time-to-contraceptive after childbirth by women aged 15-49... [/fig_ref].
Goodness of fit of the final model. We used the Cox-Snell residuals plot to check the overall goodness of fit for the final model. As depicted in [fig_ref] Fig 3: Cumulative hazard of Cox-Snell residuals [/fig_ref] , the plot of the residuals of the fitted model is fairly closer to the 45 0 straight line of the origin with slight variability in the right-hand tail, indicating that this model had a better fit to the data. Note that some variability about the 45˚line is expected even with well-fitted survival models, particularly in the right-hand tail, because of the reduced effective sample caused by prior failures and censoring [fig_ref] Fig 3: Cumulative hazard of Cox-Snell residuals [/fig_ref].
## Multivariable survival analysis result
After controlling for other factors, the use of maternal health care services, and educational status of the women were found to be the sole predictors of postpartum modern contraceptive methods use [fig_ref] Table 3: Results of Multivariable analysis of time-to-contraceptive after childbirth by women aged 15-49... [/fig_ref].
Our findings show that the hazard of postpartum modern contraceptive use was about 59% (aHR = 1.59; 95% CI: 1.22-2.06) higher for women who had a history of at least four antenatal visits compared with those who had less visits. Similarly, women who made their first antenatal visit within the first trimester had a two folds (aHR = 2.03; 95% CI: 1.28-3.21) risk of postpartum modern contraceptive use compared with those who first appeared in the last trimester.
The risk of starting modern contraceptive in the extended postpartum period was significantly higher (aHR = 1.39; 95% CI: 1.12-1.73) for women who received postnatal care within the first three days after birth compared with women who initiated postpartum care after three days.
Furthermore, in this study maternal education was significantly associated with the risk of using modern contraception in the postpartum period. When women completed primary education, their risk of using a modern method of contraceptive significantly increased by 1.30 times (aHR = 1.30; 95% CI: 1.02 1.66) compared with women who did not attend any formal education.
However, in this multivariable analysis, no statistical difference was observed in contraceptive uptake between women of different wealth status, area of residence, mode of delivery, and the number of health facilities per total population (facility density) in the district [fig_ref] Table 3: Results of Multivariable analysis of time-to-contraceptive after childbirth by women aged 15-49... [/fig_ref].
# Discussion
This study has investigated the association of socioeconomic, demographic, and environmental factors with the likelihood of postpartum contraceptive use by accounting for gamma-distributed shared frailties at cluster-level.
In this study, we used kebele as a clustering (frailty) effect on modeling the determinants of time-to-contraception after birth. The statistically significant effect of the frailty terms between different clusters revealed that the observed covariates included in the analysis were not able to account for all the variability in women's survivorship. We postulate this variability to be the combined effect of various factors that cannot be easily measured or observed at community or health facility levels including culture and social norms [bib_ref] Enhancing contraceptive choice for postpartum women in sub-Saharan Africa with the progesterone..., Ramarao [/bib_ref]. The significant level of frailty terms in our study might have biased our results if we had not taken them into account. Our interpretation, therefore, was based on the shared survival frailty model that accounts for the heterogeneity.
In this study, modern contraceptive practice during the extended postpartum period was found to be 59.5% (95% CI: 56.8-62.2%). The finding is comparable with the finding of the study done in Debre Tabor town, Northwest Ethiopia 63.0% (95% CI: 59%; 67.4%) [bib_ref] Prevalence of post partum modern family planning utilization and associated factors among..., Taye [/bib_ref]. This result was, however, higher than the 31.7% prevalence in Southern Ethiopia [bib_ref] Timely initiation of postpartum contraceptive utilization and associated factors among women of..., Dona [/bib_ref] , 45.4% in western Ethiopia [bib_ref] Role of antenatal and postnatal care in contraceptive use during postpartum period..., Teka [/bib_ref] , and 29.3% in Northern Ethiopia [bib_ref] Level and Correlates of Unmet Need of Contraception among Women in Extended..., Embafrash [/bib_ref] , and lower when compared to the studies done in Hosana town (72.9%), Addis Ababa, Ethiopia (80.3%), and 86.3% of Kenya [bib_ref] Postpartum modern contraceptive use and associated factors in Hossana town, Gejo [/bib_ref] [bib_ref] Determinants of contraceptive use among postpartum women in a county hospital in..., Jalang'o R [/bib_ref]. The low level of contraceptive use found in this study might reflect the over-reliance of lactating women on breastfeeding and menstruation status. It is evidenced that these group of women oftentimes do not realize that they are at risk of pregnancy when they are amenorrheic or breastfeeding [bib_ref] Enhancing contraceptive choice for postpartum women in sub-Saharan Africa with the progesterone..., Ramarao [/bib_ref] [bib_ref] Timely initiation of postpartum contraceptive utilization and associated factors among women of..., Dona [/bib_ref] [bib_ref] Prevalence of post partum modern family planning utilization and associated factors among..., Taye [/bib_ref]. Though breastfeeding is universal in Ethiopia, and exclusive breastfeeding up to 6 months after birth is an important contraceptive method which is highly recommended by the Ministry of Health of Ethiopia, the status of exclusive breastfeeding in the country is less than the global recommendations [bib_ref] Exclusive breastfeeding practice in Ethiopia and its association with antenatal care and..., Alebel [/bib_ref].
The main finding of this analysis is that women who started using modern contraceptive methods during the extended postpartum period were characterized by high coverage (four or more visits) and proper timing (first visit in first trimester) of antenatal care. Yet, the results of studies done in Ethiopia and elsewhere [bib_ref] Association between skilled maternal healthcare and postpartum contraceptive use in Ethiopia, Tessema [/bib_ref] [bib_ref] Postpartum modern contraceptive use and associated factors in Hossana town, Gejo [/bib_ref] showed that postpartum use of modern contraception was not affected by antenatal care. The variation could be attributed to the difference in the study design; whereas our study accounted for the hierarchical structure and tried to adjust for individual and community characteristics, the other studies were done using flat models that inherently assume the population to be homogeneous. It is evidenced that frailty models offer unobserved heterogeneity into models for survival data as random effects.
Nonetheless, the observed association between prenatal care and contraceptive use is not unique to this study and has been reported in earlier studies from Ethiopia [bib_ref] Timely initiation of postpartum contraceptive utilization and associated factors among women of..., Dona [/bib_ref] [bib_ref] Role of antenatal and postnatal care in contraceptive use during postpartum period..., Teka [/bib_ref] , and other countries [bib_ref] Postpartum contraceptive use and unmet need for family planning in five low-income..., Pasha [/bib_ref]. These studies showed a dose-response type of relationship between antenatal care and postpartum contraception adoption; that is the likelihood of using postpartum contraception increased when women had frequent antenatal contacts. Our result also demonstrated a significant association between postpartum contraceptive use and early postnatal care, which is in agreement with other studies [bib_ref] Timely initiation of postpartum contraceptive utilization and associated factors among women of..., Dona [/bib_ref] [bib_ref] Role of antenatal and postnatal care in contraceptive use during postpartum period..., Teka [/bib_ref].
The positive effects of maternity services on contraceptive uptake might be explained due to the effect of the counseling sessions and promotional efforts made during each visit. It has been indicated that each maternity services improve clients' relationships with health workers and their familiarity with the health care systems [bib_ref] The role of quality health services and discussion about birth spacing in..., Tappis [/bib_ref]. Besides, counseling and information can help women avoid social barriers and, in turn, encourage them to use health services in the future. Therefore, cognizant of the fact that only a few Ethiopian women have gotten antenatal and early postnatal services, there is a strong need to promote programs that target women who do not get these services as a strategy to promote postpartum modern contraceptive use.
There are inconsistent pieces of evidence in the correlation between women's education and postpartum contraception adoption. A study done in Northwest Ethiopia, for example, did not show any association between female education and postpartum contraceptive uptake [bib_ref] Association between skilled maternal healthcare and postpartum contraceptive use in Ethiopia, Tessema [/bib_ref]. On the other hand, in line with the previous researches [bib_ref] Enhancing contraceptive choice for postpartum women in sub-Saharan Africa with the progesterone..., Ramarao [/bib_ref] , the result of our study also confirms that educated women have a higher hazard of contraceptive use when compared to mothers with no formal education. Women's education could impact modern contraceptive uptake in different mechanisms: improving access to contraceptive alternatives, and helping them in understanding the health benefits of the available contraceptive commodities [bib_ref] Determinants of low family planning use and high unmet need in Butajira..., Mekonnen [/bib_ref] might be among the possible reasons. Education might also improve the bargaining power of women to negotiate sex, and their ability to make their own decisions, including fertilities desires [bib_ref] The effects of women's education on maternal health: Evidence from Peru, Weitzman [/bib_ref].
Results from various studies have found conflict of evidence on the link between household wealth status and the use of postpartum contraception; in some settings, it appears to be associated with contraceptive use; Hounton and colleagues, for example, reported financial constraint as a barrier to adopt postpartum contraception. However, no statistically significant difference was observed in contraceptive uptake between women of different wealth status in our study, which is in line with the results of some other studies [bib_ref] Enhancing contraceptive choice for postpartum women in sub-Saharan Africa with the progesterone..., Ramarao [/bib_ref] [bib_ref] Postpartum modern contraceptive use and associated factors in Hossana town, Gejo [/bib_ref].
This lack of variation in contraceptive use by wealth status in our study might be attributed to the introduction of healthcare financing reforms by the government of Ethiopia, which includes social and community based health insurance schemes, and charge free maternity services in public health facilities, among others. Concerning this, Dzakpasu et al reported that poor women were unwilling to use the formal health sector if they must pay for maternal health services. The Health Extension Program in Ethiopia, which brought family planning services to the community where they live, might be another reason for the lack of variation between rich and poor women. Health extension workers are deployed in pairs, two for every kebele, and affiliated with each kebele's health post to provide key health services at a community level, including family planning services since launched in 2003.
Despite we tried to estimate unbiased parameter estimates after accounting for the frailty effect, the study results should also be interpreted in light of certain limitations. The reliability of this study depends on the mother's recall of past events regarding the processes of maternal health care and therefore may be subject to recall bias. In addition, the study focused merely on the health coverage of maternal services as main predictors, yet coverage alone might not be a warranty for postpartum contraceptive use if quality was insufficient.
# Conclusions
In conclusion, the use of postpartum modern contraception was low despite the provision of charge-free services in all public health facilities. Postpartum modern contraception use was associated with increased coverage of the key maternal services, particularly the antenatal and postnatal cares. The observed strong effect of antenatal and early postnatal services strengthens the argument that integrating the key maternity could enhance the use of postpartum modern contraception. Moreover, the significant level of variance of unobserved community effect also underscores the importance of disaggregated data for evidence-based policymaking and program designing in the study area in particular and the country in general.
## Supporting information
## S1
[fig] Fig 1: Kaplan Meier survival function curve showing time to modern contraceptive use after birth among reproductiveage women in West Gojjam Zone, Northwest Ethiopia, and June 2018. https://doi.org/10.1371/journal.pone.0228678.g001 [/fig]
[fig] Fig 2: Kaplan Meier estimate curve of postpartum family planning use within 12 months from childbirth by selected characteristics of women, Northwest Ethiopia, June 2018. https://doi.org/10.1371/journal.pone.0228678.g002 [/fig]
[fig] Fig 3: Cumulative hazard of Cox-Snell residuals. https://doi.org/10.1371/journal.pone.0228678.g003 [/fig]
[table] Table 1: Background characteristics of postpartum women in West Gojjam Zone, Northwest Ethiopia, 2018(n = 1281). [/table]
[table] Table 2: Comparison of fitness of different parametric frailty models based on the Akaike information criteria, June 2018. [/table]
[table] Table 3: Results of Multivariable analysis of time-to-contraceptive after childbirth by women aged 15-49 who had their most recent birth within 12 months preceding the survey, West Gojjam Zone, Ethiopia, June 2018. [/table]
[table] Table: Data collection tool (English). (PDF) S2 Table. Data collection tool (Amharic). (PDF) [/table]
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